SARS-CoV-2 (or simply COVID-19) remains to be a global pandemic issue affecting millions, thus urging the world’s scientific community to develop efficient vaccine and design adequate measures of disease control. Currently, the most economically viable solution to infections and viruses is vaccination, despite the possible concerns about side effects from implementing quickly developed vaccine. The current commentary intends to explain the health and safety related to COVID-19 vaccines via a prism of global health safety. Scientists across the globe, along with companies from both public and private sectors, have predictably arranged cooperative programs to learn about COVID-19, along with taking simultaneous steps on devising vaccine and preparing effective treatments plans. Presently, several clinical trials to approve the efficiency of proposed vaccine solutions have been made successfully. Global health safety concerns on vaccine’s efficiency such as high costs of production, provision of vaccine to developing countries, and its influence on the global economy are addressed. This commentary reflects on current efforts related to the development of vaccine against COVID-19, which currently affects the global health status and economy. In addition, the commentary aims at addressing potential risks related to the development of COVID-19 vaccine from the global health safety perspective. The new coronavirus, also widely known as COVID-19, presumably came from Wuhan, China, starting in 2019. Wild bats were identified as potential carriers of the virus. Across generations of species, the virus eventually infected humans, spreading from individual to individual as an airborne virus. Historically, over the two last decades, there have been three major waves of coronavirus outbreaks initially transmitted from lower-order vertebrate: SARS CoV lasted between 2002 and 2003; MERS-CoV started and ended in 2012, and SARS-CoV-2, emerged in 2019 and is still prevalent in the present day. The preservation of various coronaviruses in wild bats ) as well as occasional transmissions across species that eventually affect humans beings indicates that similar cases of global outbreaks may occur in the future [After the initial outbreak in November 2019, COVID-19 has managed to penetrate 188 countries and 25 jurisdictions around the world. The governments and the WHO have been taking measures on contain the disease spread. However, the highly infectious essence of COVID-19 makes it particularly complicated. As per11:15 am CET 118,058,503 COVID-19 cases have been confirmed globally. In addition, 2,621,046 deaths have been recorded by the World Health (WHO). Moreover, 300,002,228 doses of vaccine had been administered as of 9 March 2021 [Undoubtedly, the world community has an exclusive need in access safe and valid vaccines to improve the immunity of millions of potential victims of COVID-19 and reduce the global rates of morbidity and mortality caused by SARS-CoV-2. The broad geography of impact along with the global society’s need for efficient vaccine would definitely require multi-step strategy on developing a truly valid solution. Cooperative alliances between biotechnological researchers and pharmaceutical agencies specialized in vaccine production will be critically important during the pandemic . The comThe commentary will reflect on the topic of the global health safety of the current COVID-19 vaccines by addressing three critical elements: (1) risks associated with vaccine studies; (2) vaccine production and its efficiency; and (3) using neutralizing antibodies (nAbs) produced from individuals who have recovered from coronavirus and perspectives of this practice.The recent emergence and spread of COVID-19 have underlined the necessity of developing-vaccines to fight emerging infectious diseases (EIDs). The problem is that vaccine producers have been negatively affected by the high cost of research and development (R&D) projects, research-related risks, and EID-vaccines have gained a lot of recognition in pandemic preparedness. In turn, officials and nonprofit organizations found themselves restricted in ensuring timely responses to these problems, eventually influencing overall production and regular logistics of vaccines . TherefoFrom the current limited financial perspectives, the expected economic returns from developing EID vaccine seems to be negative in relation to the cost invested. This means that the private sector alone will be unable to meet the global need for efficient vaccines without assistance from the public domain. The financial investment into an EID vaccine and realization of possible solutions are affected by increased price. Therefore, improved cooperation between public and private sectors, along with subscription options for receiving annual individual donations for accessing the list of available vaccines will be decisive .Despite the assumption that current technologies would secure quick solutions against global infections, development costs are still of critical significance , especiaEven though COVID-19 causes mainly mild symptoms in many infected people, there are individuals who seriously suffer from the disease. In selected cases, the virus can lead to lethal outcomes. The most vulnerable populations remain older people and people with preexisting and chronic medical complications, including cardiovascular problems, pulmonary complications, diabetes, and others ,7.Defining at-risk populations and profiles of victims is a crucial work that predetermines administrative decision-making and predicts the efficiency and outcomes of potential COVID-19 vaccine programs ,6. The sScientific efforts have been mainly targeted on the production of vaccines to fight COVID-19 for the purpose of reducing the pandemic’s effects. To date, a vast number of produced vaccine options have applied the S-protein taken from SARS-CoV-2 ,11,12. BAll development programs aim at producing a vaccine that can enable the synthesis of S-protein neutralizing antibodies in the bodies of vaccinated COVID-19 individuals. Research has identified that there is either a restricted or zero cross-neutralization when comparing the serum of both SARS-CoV and SARS-CoV-2. This implies that successful recovery from one disease is not a guarantee that a subject will be safe from other infectious disease . In addiCurrently, there are several COVID-19 candidate vaccines in Stage III trials. These vaccines are based on an mRNA spike protein carried via lipidic microparticles. In addition, two of the vaccines have gone beyond Stage III trials and have been authorized by WHO. On 31 December 2020, WHO approved the Comirnaty mRNAbased vaccine for emergencies, making the BioNTech/ Pfizer’s vaccine the introductory one to gain emergency authorization from WHO after the outbreak started in 2020 . The mRNIn accordance with the last WHO’s updated data, the mRNA vaccine from Pfizer-BioNTech is more effective and safer. Nonetheless, there are particular people for whom vaccines are not recommended as a result of contradictions or limited data. Presently, these individuals include the majority of pregnant women, people having severe allergies’ history, and international travellers not included in a prioritized group as well as children below 16 years. Health workers have been prioritized in vaccination efforts due to their high exposure risk, followed by the older adults, and then immunize the larger population .Other potential vaccines presently in Stage III trials are hinged on spike protein’s DNA carried through adenoviruses. Such vaccines include Russia’s Gamaley Res. Inst. Sputnik and China’s Can Sino Ad5-nCoV vaccines that have obtained limited approval and have vaccinated partial populations. Moreover, in spite of the preliminary outcomes as well as well-recorded immunogenicity, the Stage III trial of the University of Oxford/ AstraZeneca ChAdOx1 vaccine offered provocative although contradictory outcomes that need further studies.On 15 February 2021, the World Health Organization authorized two Oxford/ AstraZeneca’s COVID-19 vaccine’s versions known as ChAdOx1-S for emergencies, allowing their roll out worldwide through COVAX [On 27 February 2021, the Johnson & Johnson vaccine was also approved for emergency usage, which made it the third COVID-19 vaccine from the United States (US). Additionally, it was the first to show effectiveness and safety after receiving a single dose as opposed to two doses for the other vaccines. Furthermore, clinical trials have indicated a 72 % efficacy rate for one dose [Generally, even though these COVID-19 vaccines are promising in the prevention of deaths and severity of infections, they cannot offer 100% protection. In addition, the whole immunity process normally takes approximately 2 weeks following a vaccine’s second dosage. That is the reason why individuals can still get infected with the new COVID-19 and become sick in case of exposure shortly after receiving the vaccines [Some research projects focusing on SARS-CoV and MERS-CoV have determined that many components  located in S-proteins might be exploited as a basis for generating nAbs. Nevertheless, RBD-directed antibodies have a better chance to eliminate infection when divergent virus strains are used. It is presumed that the RBD of SARS-CoV-2 can be used as a key target for manufacturing strong neutralizing antibodies ,22,23,24The vaccine against SARS-CoV-2 should achieve the following outcomes: to reduce adverse immunopotentiation, be appropriate for use by adults above 60 or with primary features of hypertension or diabetes, be appropriate for durable accumulation, and be suitable for adult healthcare professionals . Fifty-tCurrently, the global focus is made on eliminating and preventing the COVID-19 pandemic. The permanent search for appropriate and reliable candidates for SARS-CoV-2 vaccines is impressive. The improvement in immunological reaction may be achieved by using auxiliary agents for protein vaccines. However, the large implementation of attenuated vaccines is basically related to the potential of pathogen reactivation and its subsequent toxicity . CurrentThough some side effects of the vaccine resemble the COVID-19 symptoms, this disease cannot be triggered by the coronavirus vaccine. In addition, the vaccines are not infectious. The majority of individuals experience moderate or mild vaccine side effects within 1-2 days . NeverthFurthermore, Guillain Barre syndrome and anaphylaxis are the common side effects of vaccines. Guillain Barre syndrome represents the most severe side effect caused by vaccination . NeverthThe widespread of COVID-19 changed global health priorities and realities of approaching vaccination routine. Development of vaccine in modern disturbing times is full of pitfalls. There are several critical health safety concerns regarding production and use of coronavirus vaccines. For instance, the rapid development of the vaccine might compromise its assessment before use, increasing risks of unexpected adverse effects since the vaccine itself does not give full protection from infection. In addition, health and safety is the primary field of business of the healthcare companies involved in healthcare which have to confront the economic challenges, and their interests can be beyond securing health, unfortunately. Nevertheless, it is evident that private-public partnerships along with the efforts of the scientific community, make it possible to create efficient COVID-19 vaccines that could be useful and safe in the long-term perspective.


The COVID-19 pandemic has disrupted maternity services worldwide and imposed restrictions on societal behaviours. This national study aimed to compare obstetric intervention and pregnancy outcome rates in England during the pandemic and corresponding pre-pandemic calendar periods, and to assess whether differences in these rates varied according to ethnic and socioeconomic background.p = 0.16). Preterm birth and SGA birth rates were slightly lower during the pandemic . Slightly higher rates of obstetric intervention were observed during the pandemic . Lower rates of prolonged maternal length of stay  and maternal readmission  were observed during the pandemic period. There was some evidence that differences in the rates of preterm birth, emergency cesarean section, and unassisted vaginal birth varied according to the mother’s ethnic background but not according to her socioeconomic background. A key limitation is that multiple comparisons were made, increasing the chance of false-positive results.We conducted a national study of singleton births in English National Health Service hospitals. We compared births during the COVID-19 pandemic period (23 March 2020 to 22 February 2021) with births during the corresponding calendar period 1 year earlier. The Hospital Episode Statistics database provided administrative hospital data about maternal characteristics, obstetric inventions , and outcomes , and maternal 42-day readmission). Multi-level logistic regression models were used to compare intervention and outcome rates between the corresponding pre-pandemic and pandemic calendar periods and to test for interactions between pandemic period and ethnic and socioeconomic background. All models were adjusted for maternal characteristics including age, obstetric history, comorbidities, and COVID-19 status at birth. The study included 948,020 singleton births ; 451,727 births occurred during the defined pandemic period. Maternal characteristics were similar in the pre-pandemic and pandemic periods. Compared to the pre-pandemic period, stillbirth rates remained similar (0.36% pandemic versus 0.37% pre-pandemic, In this study, we found very small decreases in preterm birth and SGA birth rates and very small increases in induction of labour and elective and emergency cesarean section during the COVID-19 pandemic, with some evidence of a slightly different pattern of results in women from ethnic minority backgrounds. These changes in obstetric intervention rates and pregnancy outcomes may be linked to women’s behaviour, environmental exposure, changes in maternity practice, or reduced staffing levels. In a nationwide cohort study, Ipek Gurol-Urganci and colleagues study obstetric intervention rates and pregnancy outcomes in England during the first year of the COVID-19 pandemic. The COVID-19 pandemic has been associated with a reduction in the rates of preterm births and small-for-gestational-age births in some high-income settings.It is unknown how changes in high-income settings have influenced rates of obstetric interventions, and whether the pandemic has exacerbated existing inequalities in maternity care and outcomes.We conducted a national population-based study, using administrative hospital records from England to compare obstetric intervention rates and maternal outcomes during the pandemic period and the corresponding calendar period 1 year earlier.Rates of obstetric interventions were slightly higher in England during the pandemic than in the pre-pandemic period.There was some evidence of slightly lower rates of preterm birth and small-for-gestational-age birth.There were very small variations in the pattern of results according to women’s ethnic background.The COVID-19 pandemic was not associated with overall worse pregnancy outcomes in England. However, there was a higher rate of obstetric interventions, which may have been the consequence of lockdown restrictions and service interruptions.The results should be interpreted with caution. All differences were small, and many comparisons were made. During the COVID-19 pandemic, pregnant women are vulnerable to both the ‘direct effects’ of infection with SARS-CoV-2 and the ‘indirect effects’ of disruption of essential healthcare services and restrictions on social interaction. There is no evidence that pregnant women face an increased risk of contracting SARS-CoV-2 infection compared to non-pregnant women. However, there is evidence that women who experience a SARS-CoV-2 infection during pregnancy may have higher rates of maternal morbidity and mortality including preterm birth, preeclampsia, and emergency cesarean delivery; neonatal morbidity; and perinatal morbidity and mortality including stillbirth . The mosThere are reports that the impact of COVID-19 differs among ethnic and socioeconomic groups, with pregnant women from ethnic minority backgrounds more likely to be hospitalised . FurtherThe population-level impacts of the indirect effects of the pandemic on maternal and neonatal outcomes are, however, likely to be larger than direct effects of COVID-19 infection –9. A recAlmost all available reports, with the exception of national data from the US, are restricted to the first 8 months of 2020 ,10. ConsIn this study, we used population-level data from England to examine the combined direct and indirect effects of the COVID-10 pandemic on mode of delivery and pregnancy outcomes. We compared obstetric intervention and pregnancy outcome rates in England during a defined ‘pandemic period’  with those during the corresponding pre-pandemic calendar period 1 year earlier (23 March 2019 to 22 February 2020) and tested whether differences in these rates varied according to ethnic and socioeconomic background.During the pandemic period, there were major changes to healthcare delivery throughout the year . For matThis study is a national population-based study using Hospital Episode Statistics (HES) records of all inpatient admissions to National Health Service (NHS) hospitals in England . HES recThe pandemic period and the lockdown periods were defined according to the timeline of events related to the COVID-19 pandemic in England: the start of the first national lockdown (23 March 2020), the relaxation of lockdown restrictions and move to a tiered system of local restrictions (24 June 2020), the start of national measures leading to a second national lockdown (22 September 2020), and the relaxation of national lockdown restrictions (23 February 2021). We chose to handle time in this way because during the lockdown periods, there were substantial changes to care and staffing: Staff were redeployed away from maternity settings, and hospital access restrictions changed . FurtherThe cohort included all women with a record in HES of a singleton birth during the pandemic period (23 March 2020 to 22 February 2021) and the pre-pandemic period . A birth (maternity) episode was defined as any record that contained information about mode of birth in procedure fields (OPCS-4 codes: R17.1 to R25.9), the main record, or the maternity tail. Multiple births were excluded. Multiple births were defined as birth episodes with an ICD-10 code for a multiple birth (Z37.2–7) or strong evidence of a multiple birth in the maternity tail .The study outcomes included stillbirth , preterm birth (less than 37 weeks’ gestation), small for gestational age (SGA) at birth (defined as birthweight < 10th centile using population-based centile charts ), inductMaternal age was grouped into 5-year periods, with women under 20 and over 39 years being aggregated into single categories. Parity and previous cesarean section were determined using women’s previous HES hospital admission records. Ethnic background was coded using the Office for National Statistics (ONS) 2001 census categorisation for ethnicity, collapsed into 4 groups: White, South Asian, Black, and Other , as there is evidence of misclassification if more granular groups are used ,20.Information about comorbidities—including pre-existing and gestational diabetes mellitus, pre-existing hypertension, and preeclampsia—was available in the diagnosis codes in the HES records of the birth episode, with women assumed not to have these conditions if the relevant ICD-10 codes were not present. A woman was classified as having laboratory-confirmed SARS-CoV-2 infection at the time of birth if the ICD-10 code for ‘COVID-19, virus identified’ (U07.1) was recorded in the birth episode . The tesThe Index of Multiple Deprivation (IMD) was used as an overall measure of area-based deprivation. It is based on the income, education, employment, crime, and living environment in an individual’s area of residence . QuintilAll outcomes and analyses described in the Methods were prespecified, with the exception of the use of multiple imputation methods for missing data and case-mix adjustment. These modifications were done following editorial and peer review. We did not publish or pre-register an analysis plan. This study is reported as per the REporting of studies Conducted using Observational Routinely-collected Data (RECORD) guideline .The characteristics of the women in the cohort were described for each analysis period, and monthly rates of different maternal and neonatal outcomes for the entire pre-pandemic and pandemic periods were plotted. Multi-level logistic regression models were used to estimate the differences in obstetric intervention and pregnancy outcome rates between the pandemic and the corresponding pre-pandemic calendar period in terms of odds ratios (ORs) and their 95% confidence intervals (CIs). ORs larger than 1 indicate that rates are increased during the pandemic period compared to the corresponding calendar period 1 year earlier. All analyses were adjusted for maternal age, obstetric history, comorbidities , and COVID-19 status at birth, and allowed for hospital-level clustering.p-value of less than 0.05 was assumed to represent statistical significance. Stata 16  was used for all analyses.A binary variable for ethnicity  or socioeconomic background  was included, and tests for interaction were used to determine if differences in outcome rates between pandemic and corresponding pre-pandemic periods varied according to ethnic and socioeconomic background. Missing values for maternal characteristics and outcomes were imputed using chained equations to generate 10 datasets; estimates from these datasets were pooled using Rubin’s rules . A p-valThis study used routinely collected administrative hospital data that were accessed to evaluate service provision and performance, and was therefore exempt from ethical review by the NHS Health Research Authority. The use of personal data without individual consent was approved by the NHS Health Research Authority (16/CAG/0058).Neither women giving birth nor their representative organisations were explicitly consulted regarding this study. However, the outcome measures used were developed in collaboration with the National Maternity and Perinatal Audit (NMPA) Women and Families Group, and we will act in conjunction with this group to disseminate the findings of this study.We identified 961,506 births in the English NHS during the pandemic period (23 March 2020 to 22 February 2021) and pre-pandemic period (23 March 2019 to 22 February 2020); 948,020 (98.6%) were singleton births. There was an overall 8.4% reduction in singleton births, from 493,293 in the pre-pandemic period to 451,727 in the pandemic period . The chap = 0.16), the first lockdown period , or the second lockdown period .The monthly stillbirth rates are presented in p < 0.001), the first lockdown period , and the second lockdown period .Compared with corresponding pre-pandemic figures , there wp < 0.001) and for the second lockdown period (p < 0.001), but there was no statistically significant difference during the first lockdown period .Compared with the corresponding pre-pandemic periods, SGA rates were lower for the entire pandemic period  and in the second lockdown period , but not in the first lockdown period  (p < 0.001), primarily driven by the higher rates in the second lockdown period . Instrumental births during the pandemic periods remained at similar levels to the corresponding pre-pandemic periods  ; Table 2 periods ; Table 2p < 0.001; 17.6% in the second lockdown versus 20.4% pre-pandemic, aOR 0.83, 95% CI 0.82 to 0.85, p < 0.001) and maternal readmission  during the first and second lockdown periods compared to the corresponding pre-pandemic calendar periods. During the entire pandemic period, the rates of prolonged maternal length of stay and readmission were also significantly lower than pre-pandemic rates . There was a greater increase in the rate of emergency cesarean birth for women of ethnic minority backgrounds (19.7% pre-pandemic to 21.9% during pandemic) compared to white women . There was a greater reduction in unassisted births for women of ethnic minority backgrounds than for white women . Otherwise, the differences were similar in women from white and ethnic minority backgrounds.There was evidence that the differences in rates varied according to women’s ethnic background for preterm births, cesarean births, and unassisted births , althougp-values for interaction > 0.05; There was no evidence that the differences in intervention rates and pregnancy outcomes varied according to socioeconomic background  , over haThis study considers interventions and outcomes at the time of birth that may have been affected by circumstances at different points throughout pregnancy. For example, fetal growth (measured as the proportion of babies born SGA) will reflect not only the impact of COVID-19 restrictions and clinical care experienced during the entire antenatal period, but also guidance by national maternity initiatives to reduce preterm birth and stillbirth –31. In aThe reduction in preterm birth rate during the pandemic period was also demonstrated in an earlier UK-based study reporting neonatal admission rates . PossiblDuring the pandemic period, our study population experienced higher rates of interventions including induction of labour and elective and emergency cesarean birth. To the best of our knowledge, similar findings have not been reported by other studies –40. AlthOur results show a reduction in prolonged maternal hospital stays, a finding supported by other studies –46; earlThere is evidence from other clinical areas that people with ethnic minority backgrounds and those from more deprived communities were disproportionately affected by the COVID-19 pandemic ,47,48. OThe main strengths of this study are its large size, with the study including almost all births in England over a period of almost 2 years, covering periods of high COVID-19 prevalence in the UK and capturing significant events along the pandemic timeline. HES data are well-established and have previously been used for national comparisons of maternity care . The cliThis study reports the differences in the rates of a number of interventions and outcomes between pandemic and pre-pandemic periods, also exploring whether these differences varied according to ethnic and socioeconomic background. These multiple comparisons, increasing the chance of false-positive results, should be taken into account, especially when interpreting small but statistically significant differences .Our study raises questions about the potential associations between societal and care changes during the COVID-19 pandemic and maternal and perinatal outcomes. The pandemic can be regarded as a natural experiment in which many aspects of care and behaviour changed simultaneously. Our results suggest that these changes may have led to reductions in the rate of preterm and SGA birth as well as increases in obstetric intervention rates. All this requires further investigation, particularly as similar results have not, to the best of our knowledge, been seen in other high-income settings .In this study of all births in the English NHS, we found a small decrease in the rates of preterm and SGA birth during the pandemic, compared to the corresponding pre-pandemic calendar period 1 year earlier. There was a small increase in the rates of obstetric interventions, which may be a consequence of temporary closure of midwife-led birth settings or a response to delays in pregnant women seeking care. These results varied slightly according to women’s ethnic background, but not according to their socioeconomic background. All these findings should be interpreted with caution, because differences were small, many comparisons were made, and the relationships between the factors influencing maternity care and pregnancy outcomes during the COVID-19 pandemic are complex.S1 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableTable A: Maternal characteristics. Table B: Maternal and perinatal outcomes.(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


As a result, they have inadequate translational value and can be poorly predictive of clinical outcome. Thus, there is a need for robust in vitro preclinical tools that more faithfully recapitulate human solid tumours to test novel immunotherapies. Microfluidics and lab-on-a-chip technologies offer opportunities, especially when performing mechanistic studies, to understand the role of the TME in immunotherapy, and to expand the experimental throughput when using patient-derived tissue through its miniaturization capabilities. This review first introduces the basic concepts of immunotherapy, presents the current preclinical approaches used in immuno-oncology for solid tumours and then discusses the underlying challenges. We provide a rationale for using microfluidic-based approaches, highlighting the most recent microfluidic technologies and methodologies that have been used for studying cancer–immune cell interactions and testing the efficacy of immunotherapies in solid tumours. Ultimately, we discuss achievements and limitations of the technology, commenting on potential directions for incorporating microfluidic technologies in future immunotherapy studies.Immunotherapy is a powerful and targeted cancer treatment that exploits the body's immune system to attack and eliminate cancerous cells. This form of therapy presents the possibility of long-term control and prevention of recurrence due to the memory capabilities of the immune system. Various immunotherapies are successful in treating haematological malignancies and have dramatically improved outcomes in melanoma. However, tackling other solid tumours is more challenging, mostly because of the immunosuppressive tumour microenvironment (TME). Current  Immunotherapy is a powerful and targeted cancer treatment that exploits the body's immune system to attack and eliminate cancerous cells. In contrast to conventional anti-cancer therapies, which do not distinguish between healthy and cancerous cells, immunotherapy can sometimes offer specific cancer cell killing and prevention against recurrence due to the memory capabilities of the immune system.2 However, only a fraction of cancer patients benefit from the current repertoire of immunotherapies.3 As a result, researchers are left with the challenge of enhancing the effectiveness of existing immunotherapies, identifying predictive markers, and discovering new immune pathways for intervention. For these endeavours to succeed, better preclinical model systems that can guide personalized immunotherapy treatments are required.Immuno-oncology (I/O) is defined as the study and development of therapies that exploit the immune system to fight cancer.4 By allowing greater control of fluid volumes, culture conditions, surface chemistry and stimuli exposure, the unique characteristics of the technology make microfluidics an ideal platform for the development and testing of immunotherapeutic agents.5–7 Microfluidic technologies are often used to test anti-cancer therapies on liquid,8,9 and solid tumours, such as spheroids, organoids10 and tumour tissue slices.11 They are also used to study immune cell behaviour, interaction and migration in a reconstructed tumour model, but have not been extensively used for efficacy studies of cancer immunotherapy. Thus, microfluidic technologies offer an underutilized resource for I/O.Solid tumours and their microenvironment, including hypoxic conditions, acidic pH, nutrient starvation, dysfunctional vasculature and the occurrence of immunosuppressive mechanisms, present significant challenges for immune cells. Microfluidic technologies can recapitulate many of these environmental features, offering a versatile tool to study solid tumour immunotherapy and representing valuable pre-clinical modelling systems.in vivo or with standard in vitro off-chip techniques. We first present an overview of the present immunotherapeutic strategies for treating solid tumours and their clinical limitations. Finally, we present an outlook on future opportunities and challenges for microfluidics in the I/O field that could impact the progression of immunotherapy research.In this review, we discuss the achievements to date made by using microfluidic platforms for I/O investigations that could not have been achieved 2.Common immunotherapeutic approaches  and theiImmune checkpoint inhibitors (ICIs) are the most successful type of cancer immunotherapy to date. Immune checkpoint molecules  are an inherent, natural system to regulate the magnitude of immune response.12 ICIs prevent the interaction between checkpoint molecules expressed on T cells and their ligands expressed on antigen-presenting cells or cancer cells. This inhibition unleashes greater T cell killing of cancer cells. Two of the most well-studied checkpoint molecules include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1).13 Ipilimumab, which targets CTLA-4, was the first ICI licensed for use in cancer patients, in the treatment of metastatic melanoma.14 The first two PD-1 ICIs to be approved were pembrolizumab and nivolumab, which show similar efficacy and longstanding results as ipilimumab. These drugs are effective in more than 25 types of solid cancers and multiple liquid malignancies.14,15 However, response rates to anti-CTLA-4 and anti-PD-1 therapies vary by cancer type with upper limits reaching only 40% in melanoma and lung cancer. Further improvements to ICIs could benefit a great number of cancer patients.Adoptive cell transfer (ACT) therapy involves the administration of immune cells to a patient. These cells can be allogeneic (taken from a donor) or autologous cells (taken from the patients' own immune cells) that have been isolated and subsequently modified and expanded ex vivo, before being returned to the patient.16,17 ACT products include tumour-infiltrating T cells, T cell receptor (TCR)-transduced T cells, chimeric antigen receptor (CAR)-T cells, NK cells and CAR-NK cells. Thus far, ACT products have had success in treating melanoma, head & neck cancer, renal cell carcinoma, gynaecological cancers as well as leukaemia and lymphomas.14 NK cell-based immunotherapy has been mostly investigated for haematological malignancies, such as acute myeloid leukemia (AML) and acute lymphoblastic leukaemia (ALL). It has shown limited success in multiple solid malignancies due to a lack of tumour infiltration and proliferation.14 In addition to the immunosuppressive tumour microenvironment limiting immune cell infiltration, acquired mutations by cancer cells may provide additional escape mechanisms to prevent T cell and NK cell attack.18 CAR-T cells are engineered to express a receptor specific to an antigen on the surface of cancer cells, such as CD19. These cells are used in leukemia and lymphoma patients,17 and they can stay active for up to a decade once injected, potentially being a one-time therapy.13 Tisagenlecleucel and axicabtagene-ciloleucel were the first to be approved for all and certain types of large B-cell lymphoma.15 For solid tumours, CAR-T cells are still being trialled in such cancers as lung, brain, breast, pancreatic, sarcoma and metastatic colon cancer.14 However, a lack of suitable cancer-specific antigens is a major limitation in the CAR-T cell field.3,15 Other challenges for CAR-T therapy include the laborious, expensive and time-consuming production.13 In solid tumours, CAR-T can be ineffective and fail to persist; although, there are examples of progress in this area, such as CAR-T cells targeting EGFR-expressing glioblastoma.13Therapeutic vaccines consist of recombinant viral, bacterial and yeast vectors, immunogenic peptides, immune cells or killed tumour cells, which are used to improve immune system activation against cancer cells.13,19–22 Cancer vaccines stimulate dendritic cell function resulting in greater T cell responses. This said, there has been little clinical success for cancer vaccines thus far when administered alone. However, promising results have been obtained when used in combination with checkpoint inhibition.14 The Bacillus Calmette–Guérin (BCG) vaccine is a nonspecific immune stimulant that was the first immunotherapy to receive FDA approval in 1990 to treat bladder cancer and remains the sole intravesical agent to prevent non-muscle invasive bladder cancer (NMIBC) progression.23 Neoantigen vaccines have shown anti-tumour benefit for melanoma and glioblastoma, whilst the TG01 mutant K-Ras peptide vaccine has been approved for pancreatic cancer.14 Dendritic cell vaccines are the most investigated vaccine type with sipuleucel-T approved in 2010 for improving the overall survival in prostate cancer patients, despite its complex production and moderate efficacy.13,19 Thus, there is room for great improvement in cancer vaccine technology.oncolytic viruses, which use the innate ability of a virus to kill cancer cells with the potential to initiate an anti-tumour response. Oncolytic viruses reproduce inside and destroy cancer cells, whilst leaving normal cells unharmed and triggering the recognition of antigens released from the lysed cancer cells that induces further anti-tumour immune responses.14 Oncolytic viruses can be beneficial in treating melanoma and brain cancer.15,18 With regard to solid tumours, intratumoural injection are ideal for direct cancer cell lysis without detrimental systemic effects or hepatic virus degradation.18 Unfortunately, this procedure is not technically possible for most metastatic cancers. Roadblocks for this form of therapy, in addition to those caused by the tumour microenvironment (TME) and in particular the dense fibrotic tissue surrounding tumours, include complete viral clearance, and acquired specific immunity against the virus, meaning that repeat therapy is not possible.14,18Another type of immunotherapy involves cytokine therapy may be employed. Cytokines, such as interferons, interleukins, and granulocyte-macrophage colony-stimulating factor (GM-CSF), can boost T cell responses, activate apoptosis programs in cancer cells, delay angiogenesis, stimulate dendritic cell maturation, and slow cancer progression.13,18,24 A disadvantage to the use of cytokines, however, is their short half-life. This means that treatment needs to be delivered at a high dose, which can result in serious side effects, such as cytokine release syndrome (CRS), vascular leak syndrome and autoimmune attacks.13To stimulate anti-tumour immunity in cancer patients, 25 The STING pathway, which is a cytoplasmic DNA-sensing system , represents another area of active research for immunotherapy, because it activates anti-cancer immune responses. The STING agonist, ADU-S100, benefits patients with triple negative breast cancer and melanoma when given in combination with spartalizumab, a checkpoint inhibitor.26For many immunotherapies to be successful, immunosuppression must be overcome and anti-tumour immunity must be engaged. This may be accomplished by targeting immunosuppressive enzymes, such as indoleamine-2,3-dioxygenase (IDO1) and arginase.in vitro models have been established to investigate the effects of therapeutic agents,27 with reports of immune cells being unable to kill cancer cells in a 3D environment, despite doing so in 2D assays,28 or with anti-tumour effects of T cells being significantly reduced in 3D cultures.29 The complexity originating from the intricate spatial cellular organizations within the TME and their interactions with immune inhibitory mechanisms highlights the need for complex in vitro models that faithfully mimic human in vivo scenarios.30,31 Various solutions to these challenges can be provided by microfluidic and organ-on-a-chip technologies that enable miniaturisation, maximise the use of intact human tumour tissue, allow the robust and controlled modelling of tumour microenvironment characteristics and facilitate the culture of multiple cell types in defined spatial and temporal configurations.27 The application of these technologies in the field of immune-oncology is discussed in more detail below.Targeting solid tumours with immunotherapies is a field in development. Many 2D and 3D 3.in vitro models to be formed and easily studied with a variety of microscopy techniques. This is achieved more easily than for in vivo models, because they allow manipulation of specific experimental variables for mechanistic studies.32,27 Whilst the technology has been used to develop I/O assays for liquid tumours,6 its use in studying solid tumours has been less frequent.7,29 The approaches used to date have been grouped below according to their application in immune-oncology.Microfluidic systems provide a cost-effective and sample-efficient platform that enables complex Several studies have focussed on investigating the interactions between cancer and immune cells .et al. developed a microfluidic device to study the role of the IRF-8 transcription factor in cross-talk between murine melanoma and immune cells and the release of soluble factors resulting in immune cell infiltration.36 The device was composed of an enclosed central microfluidic chamber with externally accessible compartments on either side for culturing adherent B16.F10 murine melanoma cells and non-adherent splenocytes from wild type (WT) and IRF-8 KO mice. Compartments were connected by an array of microchannels that permitted chemical and physical contact amongst the two cell types. It was observed that tumour cells were more invasive when cultured in vitro alongside IRF-8 KO splenocytes rather than WT splenocytes. Consistently with in vivo findings, this suggests IRF-8 inhibits tumour cell invasion. A previous study by Businaro et al. also revealed, through FACS analysis, an upregulation of CD69, a marker for activation, for WT splenocytes.34 Further investigating the interaction between WT/IRF-8 KO immune cells and murine B16.F10 melanoma cells, Agliari et al. created mathematical models and performed data analysis on the dynamic movement of the splenocytes.35 More recently, Biselli et al. utilised the same platform to study interactions between doxorubicin treated human breast (MDA-MB-231) and colon cancer cells (patient derived and HCT116 cell line) and human PBMC  expressing different genetic variants of the FPR1 gene, known to influence the response of phagocytic cells  cancer cells exposed to decitabine (DAC) and/or IFN.42 Greater infiltration of PBMC was reported toward tumour cells treated with both DAC and IFN in comparison to one agent alone and to untreated cells.Mattei ic cells .41 PBMCset al. developed a multi-channel setup where human umbilical vein endothelial cells (HUVEC) were co-cultured with hydrogel-embedded tumour aggregates and macrophages.37 The effect of different macrophage subtypes on tumour aggregate dispersion was studied to mimic epithelial–mesenchymal transition (EMT), inducing cancer cell dissociation via CD11b and ICAM-1 interaction. The study showed ICAM-1 involvement in the dissociation and migration of tumour cell aggregates. The use of microfluidics in this application allowed real-time monitoring and precise measurements of cell–cell distances. Subsequently, Chen et al. studied the role of neutrophils in promoting cancer cell metastasis under perfusion conditions in order to assess their potential as targets for cancer immunotherapies . Results indicated that lactate was able to reprogram macrophages from pro-tumour cells to anti-tumour cells and to reduce cancer cell viability when in co-culture with macrophages. The quantitative and dynamic observation of macrophage migration and behaviour could not have been achieved with conventional methods, such as tube formatting assays and transwell migration assays. A similar device was also used for co-culturing four different cell types to depict a bladder cancer microenvironment using perfusion equipment to deliver continuous flow (39 The system was composed of a U-shaped channel with ECM (Matrigel) infused media and cell culture channels interconnected by microchannels to allow the different cell types to exchange soluble biological factors and metabolites. The sensitivity of cancer cells to six different chemotherapy regimens was assessed to study in vitro the effects on macrophage migration to the tumour compartment.A microfluidic co-culture chip, consisting of an ECM filled channel (Matrigel) and seven migration channels between culture chambers, was created to study the role of lactate on macrophage recruitment and cytotoxicity against cancer cells, which has relevance to BCG immunotherapy.ous flow .39 The set al.46 A device was designed to test the potential of chemokine-loaded NPs to elicit the migration of DC and T cells through an endothelial barrier towards cancer cells. Quantification of DC and T cells showed a greater presence of these cells after administration of chemokine-loaded folic-acid NP in comparison to folic-acid NP only.The role of nanoparticles (NPs) in immunotherapeutic cell interactions has also been investigated. Chemokine-loaded folic-acid conjugated NPs targeting folic-acid receptor expressing cancer cells were studied in a work by Wimalachandra 47 The device was developed to better understand the interactions between cancer and immune cells involved in tumour escape from immune surveillance. The chip allowed study of crosstalk between different cell types which is difficult using traditional Petri-dishes or well-plates. It can also be used to carry out neutralising and competitive assays. Paper-based microfluidic devices are cost-effective and can maintain oxygen and nutrient gradients that depict organ-level functions.Alternative device materials have also been used for immunotherapy studies in microfluidics. A novel microfluidic device was made of a paper layer containing five microreactors on top of a PMMA layer with hydrogel diffusion channels .47 The dThe most commonly studied form of immunotherapy using microfluidic technology is ACT therapy .48 The platform consisted of interdigitated electrode structures and integrated fully spray-coated organic photodiode arrays for electrical and optical light scattering measurements under perfusion conditions. Cellular impedance sensing was used to detect cell surface interactions while light scattering was used to determine the quantity and morphology of cells present, enabling simultaneous study of tumour cell invasion and escape of immune surveillance. A few years later, a microfluidic device was developed for tracking the migration and speed of T cells through a controlled chemokine gradient,49 showing that inhibiting the MYCN (N-Myc) proto-oncogene could improve T cell infiltration. Other work looked at the use of aminobisphosphonates (N-BPs), such as zoledronic acid (ZA), as nanoparticle agents to specifically promote anti-tumour Vδ2 T cell proliferation , human primary monocytes were used to mimic the hepatitis B virus (HBV)-related hepatocellular carcinoma tumour microenvironment .55 T celet al. designed a multi-chamber microfluidic device to assess NK cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC).53 NK cells were either embedded in hydrogel alongside cancer spheroids or perfused through endothelial channels, so that NK and antibody penetration in 3D and through the vasculature could be observed. Z-Stack images showed antibodies administered into side channels were able to extravasate from the endothelial layer and penetrate the ECM, but not the spheroids. NK cells, however, were able to home in on tumour spheroids located hundreds of microns away and to successfully infiltrate and kill tumour cells on the periphery of a spheroid, as well as in its core. More recently, the same group adapted the microfluidic set-up to investigate NK exhaustion due to tumour environmental stresses reproduced on-a-chip.59 The device design allowed to control nutrient and pH gradients across 3D tumour models, as well as inducing cell proliferation and necrosis. Gene expression analysis showed greater exhaustion of the NK cells cultured in devices in comparison to those in well plates. However, this effect could be partially negated with the use of an anti-PD-L1 antibody, atezolizumab, and IDO-1 inhibitor, epacadostat. Results demonstrated suppression of NK cell cytotoxicity for regions where MCF7 cells were able to create environmental gradients supportive of their proliferation. In contrast, patient derived breast cancer cells were unable to elicit sufficient environmental stress to be detrimental to NK cell cytotoxicity.NK cells were also recently investigated with microfluidic studies. Ayuso 54 Single cells were manipulated into PEG-DA hydrogel four-leaf-clover-shaped microwells, where direct cell-to-cell contact could occur. The benefit of this model was that it avoided continuous fluid flow and shear stress in the specific device regions where secreted proteins would be washed out and interactions between cells and NK cell activity were impacted. Park et al. proposed a 3D cytotoxicity assay to determine cytotoxic capabilities of lymphocytes -based optoelectronic tweezers was established for controlling cell–cell contacts to study immune and cancer cell interactions .54 Singlphocytes .56 PolysOther immunotherapy-related approaches have also been investigated in microfluidics .60 Greater tumour cell intravasation and endothelial permeability was reported when tumour cells were co-cultured with macrophages. This could be reduced by administering anti-TNF antibodies. A device originally used to study angiogenic growth was employed by Jenkins et al. to test a novel TBK1/IKKε inhibitor on murine-derived organotypic tumour spheroids (MDOTS) and patient-derived organotypic tumour spheroids (PDOTS).61 Greater cell death of MC38 MDOTS was seen when the inhibitor was combined with anti-PD-1 treatment and was dose- and time-dependent.A microfluidic platform has been developed and used for examining the role of macrophages in tumour cell intravasation.et al. for testing of immune checkpoint blockade (ICB) in conjunction with small hydrophobic molecules, as these can be adsorbed by PDMS.62 Murine- and patient-derived organotypic tumor spheroids from a patient with a small intestinal neuroendocrine tumour (SI-NET) were treated with ICB treatment with anti-PD-1 + anti-CTLA-4 in combination and as monotherapy. Dual ICB showed greater immune-mediated killing and relative expansion of CD8 T cells and macrophages in comparison to control and single agent ICB-treated PDOTS. Biopsy fragments were also studied in microfluidics developing an ex vivo immuno-oncology dynamic environment  in a pressure-pump-driven system. This set-up was utilised in ICB studies and reported increased tumour killing seen in channels with TILs treated with anti-PD-1 ICI. However, issues with fluid obstruction due to bubble formation and cell debris accumulation were recorded.The use of plastic microfluidic devices made using cyclic olefin polymer (COP) have also been proposed by Aref ironment .63 This et al. investigated IFNα-conditioned dendritic cells for use as a therapeutic vaccine in combinations with chemotherapy drug, romidepsin.68 Migration channels connected the tumour and immune chambers to allow analysis of DC migration, phagocytosis and cell–cell interactions, aiming to mimic in vivo DC crossing of the endothelial barrier. A cell electrofusion device that can pair and fuse homogeneous and heterogeneous cells was produced by Lu et al. and is relevant to the production of DC-tumour fusion vaccines to elicit anti-tumour immunity  in a simplified reconstructed TME, rather than looking at the effects of specific immunotherapy agents. Of the actual immunotherapy regimes tested, ACT therapies (33%) were the most studied, followed by ICB (11%), antibodies (8%), DC vaccines (6%) and cytokines/chemokines (6%). Immune cell mediated cytotoxicity has increasingly generated interest in recent years and has seen success in a range of haematological malignancies, potentially driving the development of microfluidic assays in this area. Overall, the miniaturised environment and the enhanced control over cell positioning obtained in microfluidics is inherently suitable for building advanced complex models that are not easily achievable with off-chip techniques. Therefore, it is not surprising that over two thirds of the models implemented ACT studies and cell-interaction/migration investigation, these being considerably more challenging to image Over a third of the papers considered in this review relied solely on immortalized cell lines. Over half of the studies used a combination of primary cells and cell lines, while only 8% used primary tissue alone. Although using primary tissue increases the complexity of the assays  and the cost, it does enhance their translational value. As a number of these studies were proof-of-concept work, it could be argued that using primary cells would have been an unnecessary complication, as cell lines guarantee an easier approach to illustrate the operation of the device and validate the assay initially. Nonetheless, it is essential that follow-up studies emerge showing outcomes that go beyond the scope of proving feasibility of the microfluidic technology alone.73 Within the cohort of papers reviewed, the vast majority (80%) of the devices were fabricated in polydimethylsiloxane (PDMS), with few reports of thermoplastic, paper and glass microsystems being used. Soft-lithography is abundantly used in research laboratories, this involves the fabrication of microstructures via replica moulding from master templates, most of which are created by photolithography.74 The most popular material option for this is PDMS, due to its favourable bio-chemical properties and lower cost compared to alternative materials.75 Its optical transparency allows straightforward visualisation, while its gas permeability facilitates cell culture.75,76 It can be adjusted to obtain the desired degree of elasticity, using cross-linking agents.77 The flexibility of PDMS allows for its use in a wide range of applications. However, PDMS also presents disadvantages such as bulk absorption of compounds, especially important for compound testing,73 and mass production of PDMS based devices is less straightforward than thermoplastic-based manufacturing. Glass and silicon microfluidic devices are more expensive and time consuming to process.78 However, glass is a durable material and presents less risk of drug absorption than PDMS.73 Thermoplastics, such as polycarbonate and polystyrene, can be readily oxidised to decrease their hydrophobicity for use in microfluidics,79 and are also better characterized for drug absorption compared to PDMS, but microfabrication of small features and bonding of several layers are not straightforward processes compared to soft-lithography.73 Overall, the material chosen for the device has considerable implications for the intended application of the microfluidic assay. Importantly, interfacing of microfluidic structures, harbouring 3D cellular models, with automated imaging techniques and novel image analysis algorithms can play a fundamental role in large throughput applications and in the standardization of device features.Generally speaking, microfluidic devices are fabricated using various polymeric materials, from elastomers to thermoplastics, but also paper and glass as substrates.via injection or perfusion using tubing connected to microfluidic devices. The use of cumbersome, tubing-based external instrumentation often poses challenges to the development of the technology for high throughput applications.44 Requirement for automated perfused cell culture is typically the main driver in deciding whether continuous connection to external equipment is required. Other solutions are available that can be implemented with open well-based microfluidics, such as interfacing with robotic dispensers,80,81 plate rockers82 or bespoke microfluidic layouts that can achieve long-lasting, self-generated perfusion.83,84The majority of the works under consideration used fluid actuation equipment, with 61% of the microfluidic devices relying on manual pipetting alone, whilst 20% were connected to syringe pumps to achieve flow perfusion. A further 19% of papers required a multi-equipment system. Syringe, pneumatic, or peristaltic pumps are commonly used to provide a controlled, continuous delivery of nutrients to cells via microchannel networks or hydrogel barriers. This is possibly the biggest advantage that the microfluidic mesoscale can offer with respect to other techniques. It is able to create complex in vitro multicellular models in 3D in a controlled manner, enabling steep molecular gradients to be formed, cell chemotactic mechanisms to be reproduced and different viscous fluids (i.e. media and hydrogels) to be used concurrently, recreating several TME characteristics and offering the possibility to study the interactions between various immune, stromal and tumour cell types. The miniaturised scale of microfluidic devices can also provide opportunities to perform such assays using limited patient derived samples. However, areas for improvement remain and the technology requires further development for use in assays involving a broader range of immunotherapies.Finally, yet importantly, a predominant structure emerged regarding the layout of the microfluidic device used, supporting cell interaction and migration studies. Individually addressable, parallel multi-chamber designs  and immunocompetent (i.e. presenting an intact immune system) mice. Human cancer cell lines and primary cancer patient tumour tissue can be engrafted into immunodeficient mice, but these lack adaptive immune cells and therefore may fail to fully recapitulate immune responses observed in immunocompetent cancer patients.3 Many studies have also reported issues with successful engrafting of human cells and their long-term survival in the host. While cytokines can be administered in an attempt to prolong human cell survival in these models, this has the potential to skew the immune response and could lead to the acquisition of data that is not representative of the actual human immune response.92,93 Animal studies are thought to overestimate the benefit of investigated treatments by approximately 30%, with less than 8% of in vivo assays able to be successfully translated into clinical cancer trials.93 These methods also have the disadvantage of the high costs of experiments, in addition to ethical concerns.94 Advanced in vitro I/O models could avoid many of the drawbacks of animal experimentation.The lack of in vitro models can potentially reduce and, in some cases, replace less predictive 2D assays and more expensive animal models, providing a scalable and versatile platform for immunotherapy development.95,96The adoption of microfluidic models into the pharmaceutical industry could play a significant role during drug development. Chip-based, advanced and complex 5.in vitro and animal models, improving our understanding of I/O mechanisms and creating predictive and efficient tools for the cost-effective development of immunotherapies. Looking at the next 10 years, considerable challenges have to be overcome to achieve these objectives  have the potential to offer solutions that can one day outperform current jectives .Most of the studies described in section 3 focussed on investigating mechanisms of action and represented proof-of-principle studies related to immunotherapies. Therefore, further evidence is needed to demonstrate the full potential of microfluidic assays for testing efficacy of immunotherapies. Follow-up studies are required that use the technology to drive the medical or biological investigation, which will in turn optimise the technology and define specific applications. Technology transfer and increased multidisciplinary collaborations are key aspects to achieve such aims. Alongside this is the need for funding programmes that can sustain diverse and large teams working collectively towards the common goal. Increasing model complexity and data throughput will require side-by-side advances in the automation of the microfluidic protocols, as well as in the development and application of machine learning algorithms and high-level data interpretation. As well as a broad academic team, there is a need for collaboration with Pharma industry and the clinical sector.in vivo animal experiments, with considerable advantages for straightforward interfacing to high content imaging equipment. Building upon the current microfluidic assays developed for ACT therapy and cell interaction/migration highlighted in this review, we envisage that the technology can be used to develop new assays to identify and isolate immune cells from patient derived tissue and test the efficacy and specificity of T cells (including CAR-T cells). Additionally, the technology could be used to assess the efficacy and penetration of oncolytic virus and antibodies into 3D in vitro human models. Finally, the development of body-on-a-chip platforms, where simplified systemic aspects of the immune system could be modelled, represent challenging but rewarding new areas that would benefit those immunotherapies currently under-investigated in microfluidics experiments.It is expected that successful validation of organ-on-a-chip and microfluidic technologies for a specific immune cell type will drive its use to study anti-cancer effects on the tumour in the context of various other immune cells. This will facilitate mechanistic studies at a lower cost than 97 Together with increased use of human tissue, there will be a requirement for non-animal derived hydrogels and supplements to be developed and used, as cross-species contamination could adversely affect the predictive value of the assays.Miniaturisation makes it feasible for small biopsies to be used to test multiple therapies in parallel (either using live micro-fragments or single-cell digested samples), on a scale not currently possible with macro-size tools. The resulting increased throughput of information extracted from small biopsy samples means that more samples can be tested and more quickly, without the need for long periods of cell expansion from biopsy cultures. This increases the feasibility of producing results in a clinically meaningful time-frame for personalised therapy. Moreover, the use of human clinical samples is much more likely to identify clinically relevant mechanisms of resistance than immortalised cell lines or animal models.in vitro models. Increasing success in the development and validation of new microfluidic I/O assays is expected to drive the integration of the technology first into routine R&D practise in the pharmaceutical industry and, subsequently, into clinical care for precision medicine.It is expected that over the next few years, the ability of microfluidic assays to predict clinical outcomes using patient samples will be assessed initially retrospectively and then prospectively. This will drive confidence in the technology and facilitate regulatory approval when using However, limiting factors are currently present that do not facilitate access to patient samples and collection of clinical information. New policies should be put in place to accelerate and simplify access to and sharing of patient samples and clinical/health data for research at the national and international levels. This is a key aspect to the faster development of the technology and its global use in the biotech industry. Longer term, the use of functional assays with patient-derived tissue could be a game changer in selecting effective personalised therapies for patients.i.e. Dendreon, Kite, Novartis, Aivita, Iovance Biotherapeutics, Leucid, Adaptimmune, Immunocore, Carisma and Allogene to name a few). However, the majority of associated in vitro tests are carried out in 2D or using standard well plate technologies prior to animal models. The miniaturization capabilities combined with the large data throughput of microfluidic systems offer a unique opportunity to integrate human tissue-based advanced assays into industrial preclinical studies for the development of stratified therapeutic approaches. Since microfluidics can be used for large-throughput screening, existing immunotherapies could be tested in combination with novel agents as part of a combinatorial regime.29,98 The latter is expected to become a particular area of interest as the effect of cell therapy can be enhanced when applied in combination with chemotherapies, radiotherapies or ICIs.99In the past 10 years, there has been an explosion of biotechs and Pharma investing in new programmes for developing autologous and allogeneic cell therapies (The landscape depicted in this review shows that in the field of immunotherapy for solid tumours, the use of the technology is maturing, but is still in the early stages. A coordinated effort from a variety of disciplines is required to widen the field of application, to produce clinically relevant data and to promote constructive engagement between developers and end users of new microfluidic technologies.There are no conflicts to declare.


A 48-year-old woman had an acute blurred vision in the right eye immediately after drainage of liver abscess. Her best corrected visual acuity (BCVA) was 8/400; fundus photography suggested the diagnosis of endogenous endophthalmitis with chorioretinitis and vitritis. Due to the bad systemic condition, a systemic antibiotic combined with periocular triamcinolone (TA) was carried out first. Inflammatory cells in the vitreous cavity were decreased after treatment; however, fundus fluorescein angiography (FFA) showed abnormal dilation and leakage of the capillaries and retinal-choroidal anastomose, supporting that there was retinal angiomatous proliferation (RAP). Vitreous interleukin-6 (IL-6) was only slightly elevated; the ratio of interleukin-10 (IL-10) and IL-6 was less than 1, and the etiological test was negative. After receiving intravitreal vancomycin injection combined with periocular TA injection, the patient's BCVA was improved from 16/400 to 20/400 with a reduction in vitreous inflammatory cells. However, the patient's RAP was progressed and her BCVA was dramatically decreased to count finger/30 cm. After intravitreal injection of ranibizumab, the patient's BCVA was 5/400 with a significant shrink in lesions and absorption of hemorrhage, exudation, and fluid. Thus, we suggest that early anti-inflammatory treatment in conjunction with anti-VEGF may achieve a better prognosis in patients with inflammatory retinal angiomatous proliferation (RAP). Chorioretinitis is a type of posterior uveitis. According to the etiology, it can be divided into noninfectious and infectious chorioretinitis . Early rA 48-year-old female complained of defective vision in the right eye immediately after drainage of liver abscess. Her best corrected visual acuity was 8/400 in the right eye and 20/20 in the left eye. There was no evidence of anterior chamber cells or keratic precipitates, and the intraocular pressure was normal. Dense vitreous opacities obscured the visualization of fundus of the right eye. Chorioretinitis with exudation and hemorrhage were present in the macular and inferonasal peripheral retina Figures . The funFour months later, the patient returned to the ophthalmology department with stable condition after the antibiotic therapy. Her BCVA was 16/400 and vitreous inflammatory cells in the right eye were decreased; flaky yellowish-white elevated lesions surrounded by hemorrhages can be visualized in the macular and inferonasal peripheral retina. The crooked retinal arterioles and retinal venules extended into the outer nuclear layers of the macula of the right eye . OCT shoFive months later, the inflammation was under control , but herIn this case, the patient had blurred vision immediately after drainage of liver abscess; Klebsiella pneumoniae was positive in blood culture, so endogenous infectious endophthalmitis was assumed at the initial diagnosis. Considering the poor systemic condition of the patient, systemic antibiotic therapy combined with periocular TA was carried out first . Four moIn inflammatory eye diseases, inflammatory factors promote the upregulation of vascular endothelial growth factor (VEGF), accelerate the development of abnormal angiogenesis, and often result in concurrent inflammatory CNV , 12. RAPMost of documented studies have recommended the application of intravitreal injection of anti-VEGF drug for treating RAP, which was considered as a form of exudative AMD –19. SincRAP with endophthalmitis is rare; early anti-inflammatory and anti-VEGF treatments were effective in this case. When vitreous humor and laboratory results are ambiguous to distinguish infectious from noninfectious inflammation, the history and clinical signs may help to support the primary diagnosis and experimental treatment.


SARS-CoV-2 nosocomial outbreaks in the first COVID-19 wave were likely associated with a shortage of personal protective equipment and scarce indications on control measures. Having covered these limitations, updates on current SARS-CoV-2 nosocomial outbreaks are required. We carried out an in-depth analysis of a 27-day nosocomial outbreak in a gastroenterology ward in our hospital, potentially involving 15 patients and 3 health care workers. Patients had stayed in one of three neighboring rooms in the ward. The severity of the infections in six of the cases and a high fatality rate made the clinicians suspect the possible involvement of a single virulent strain persisting in those rooms. Whole-genome sequencing (WGS) of the strains from 12 patients and 1 health care worker revealed an unexpected complexity. Five different SARS-CoV-2 strains were identified, two infecting a single patient each, ruling out their relationship with the outbreak; the remaining three strains were involved in three independent, overlapping, limited transmission clusters with three, three, and five cases. Whole-genome sequencing was key to understand the complexity of this outbreak.IMPORTANCE We report a complex epidemiological scenario of a nosocomial COVID-19 outbreak in the second wave, based on WGS analysis. Initially, standard epidemiological findings led to the assumption of a homogeneous outbreak caused by a single SARS-CoV-2 strain. The discriminatory power of WGS offered a strikingly different perspective consisting of five introductions of different strains, with only half of them causing secondary cases in three independent overlapping clusters. Our study exemplifies how complex the SARS-CoV-2 transmission in the nosocomial setting during the second COVID-19 wave occurred and leads to extending the analysis of outbreaks beyond the initial epidemiological assumptions. Similarly, genomic analysis has been applied to evaluate local SARS-CoV-2 spread and better understand its transmission dynamics during an outbreak (https://www.krisp.org.za/news.php?id=421). Outbreaks in nosocomial settings are of particular relevance, as they may affect vulnerable individuals and health care workers, who themselves may become transmission vectors and are associated with a higher risk of mortality  was observed, associated with an elevated inflammatory marker. Forty percent of the patients (6/15) received systemic corticosteroids, and 46.7% required oxygen support. Two patients were admitted to the intensive care unit (ICU) and required invasive mechanical ventilation. COVID-19-related mortality was 26.7% , 4 days [2.75 to 8.25]} of being infected. Imaging tests results and/or symptoms compatible with COVID-19 allowed making the diagnosis in 14 patients and three HCWs. The remaining case was diagnosed after screening was done due to close contact with a COVID-19 case. Following the protocol established by the hospital, a negative RT-PCR was required to be admitted to a non-COVID ward. Therefore, patients were considered confirmed or probable nosocomial infections; in five cases, the period between their last negative SARS-CoV-2 RT-PCR and a COVID diagnosis was >14 days and 3 to 13 days for the remaining 11 cases.Genomic analysis of 13 cases (12 patients and 1 HCW) for which sequencing material was available  allowed identification of five different SARS-CoV-2 strains, an unexpected diversity , left. WTwo of the strains were found each in a single patient (strains 4 and 5), ruling out the implication of these cases in the outbreak. The remaining three strains were involved in limited independent transmissions with 3, 3, and 5 cases in each cluster . The HCW (case E), initially thought to have been infected after a household exposure, was associated with cluster 1.Strain distribution among the three rooms affected by the outbreak  is shown in https://www.krisp.org.za/news.php?id=421). Additionally, nosocomial transmission increases the risk of exposure to HCWs .Some large nosocomial outbreaks occurred during the first wave of COVID-19, mainly attributed to a shortage of PPE and lack of clear guidance on prevention and control measures. Efforts have been made to increase our knowledge on the dynamics of SARS-CoV-2 nosocomial transmissions in the first wave (3–https://www.krisp.org.za/news.php?id=421). The current use of real-time genomic epidemiology has fully proved its benefits , interpreted as caused by patient-to-patient transmission and a potential role of contaminated surfaces, provided a completely different perspective.WGS revealed that five different strains were coinciding in the ward during the outbreak, three of them shared by more than one patient. This suggested independent simultaneous importations of several strains from the community, which were then subsequently nonsocomially transmitted. This hypothesis was supported by performing an integrated analysis of the sequences from the cases in the ward with representative sequences of other circulating strains from the community for the same time period.Therefore, our analysis supported that five different strains had been introduced in the ward. When the outbreak occurred, the gastroenterology ward received COVID-19-free patients from the pulmonology and internal medicine departments. Our WGS-based findings indicate that the currently applied standard measures, only addressed to reduce transmission once the patients are in the ward, are not enough if they are not accompanied by additional controls to prevent the introduction of undiagnosed presymptomatic or asymptomatic COVID-19 cases.The five introduced strains coincided in time and involved patients staying in three neighboring rooms. Despite this spatial-temporal coincidence, only half of the strains were further transmitted, and the rest did not cause any secondary cases in the ward. These findings suggest the likely existence of singular, specific factors responsible for the outbreak rather than a general major systematic control deficiency.The restriction of the outbreak to three neighboring rooms, with the sequential turnover of different strains in the cases who had occupied the rooms, minimizes the initially assumed possibility of contaminated surfaces. Although the primary transmission mode of SARS-CoV-2 in hospitals is close contact and exposure to droplets or contaminated fomites, our findings suggest airborne transmission. Air turnover was checked in the ward, and room 1 showed the lowest values of inward airflow. Room 3 was not connected to the general ventilation system to impulse air into the room; instead, a fan coil had been installed that could be manually switched off . Finally, room 2 was adjacent to the one with the lowest pressure of inward airflow (room 1), which may cause airflow distortions when doors are kept open. These data suggest that possible deficiencies in the air system may be a contributing factor for the concentration of cases in the three indicated rooms.This study shows the importance of WGS-based analysis to correctly understand the true complexity behind nosocomial transmission events. This technique provided key data to interpret the here-reported outbreak, and the involvement in the outbreak of an HCW, initially thought to have acquired the infection outside the hospital, was only understood once WGS data were available.We report a complex epidemiological scenario of a nosocomial COVID-19 outbreak in the second wave based on WGS. Initially, standard epidemiological findings led us to assume a homogeneous outbreak caused by a single SARS-CoV-2 strain, suspected to be somehow virulent due to the patients’ outcomes, driven by person-to-person transmission and contaminated surfaces. The discriminatory power of WGS offered a strikingly different perspective consisting of five importations of different strains, with only half of them causing secondary cases in three independent overlapping clusters and with a turnover of strains within the same rooms that ruled out the role of contaminated sources.This was a retrospective study in a tertiary referral hospital in Madrid (Spain) that included all consecutive patients diagnosed with COVID-19 at admission (15 September to 12 October 2020) in a non-COVID-19 gastroenterology ward, as well as the health care workers (HCWs) who were in charge of these patients and diagnosed during the study period. Baseline characteristics and clinical and laboratory parameters of the patients at COVID-19 diagnosis and their outcome were obtained from their electronic medical records. Data were analyzed with the SPSS 20.0 package . Numeric variables are expressed as medians and interquartile ranges (IQRs) and categorical variables as the number of cases and their percentages.in vitro diagnosis (CE-IVD) RT-PCR kit .Viral RNA was extracted and purified from 300 μl of nasopharyngeal exudates with the aid of the KingFisher  instrument. Next, an RT-PCR was performed, using the TaqPath COVID-19 CE-marked for https://artic.network/ncov-2019) and the Q5 Hot Start DNA polymerase . Libraries were prepared using the Nextera Flex DNA library preparation kit  following the manufacturer´s instructions.Eleven microliters of RNA were used as the template for reverse transcription using Invitrogen SuperScript IV reverse transcriptase  and random hexamers . Whole-genome amplification of the coronavirus was done with an Artic_nCov-2019_V3 panel of primers   before being pooled at equimolar concentrations (4 nM). Next, they were sequenced in pools of up to 17 libraries on the MiSeq system  and the MiSeq Reagent Micro kit v2 (2× 151 bp) or in pools of up to 96 libraries with the MiSeq reagent (2× 201 bp).FASTQ files above the Global Initiative on Sharing All Influenza Data (GISAID) thresholds were deposited at GISAID .https://github.com/pedroscampoy/covid_multianalysis. Briefly, the pipeline goes through the following steps: (i) removal of human reads with Kraken (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), (iii) mapping with bwa v0.7.17 (NC_045512.2) as reference, and (iv) recalibration of punctual low-coverage positions using joint variant calling. When necessary, informative noncovered positions were analyzed by standard Sanger sequencing with the corresponding flanking primers from the Artic set.An in-house analysis pipeline was applied to analyze the sequencing reads. The pipeline can be accessed at h Kraken , (ii) prh Kraken  . Informed consent was obtained, and consent for publication was obtained.