Removal Of Blood Cells Research Articles

Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer’s disease cortex

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer’s disease (AD). New MethodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

A hand-powered SERS-microfluidic chip for circulating tumor DNA detection from whole blood

Portable devices for rapid, automated and quantitative nucleic acid detection from whole blood is highly desirable for point-of-care (POC) molecular diagnostics. Conventional sequencing and polymerase chain reaction methods usually require trained personnel to deal with the time-consuming procedures, from sample purification to target amplification. To promote POC disease diagnosis and monitoring, we herein developed a hand-powered microfluidic chip for circulating tumor DNA (ctDNA) analysis from whole blood. On the one hand, the chip allowed rapid blood cell removal through a microfluidic filter trench. On the other hand, the ultrasensitivity of surface enhanced Raman spectroscopy (SERS) technique enabled amplification-free DNA mutation screening. Meanwhile, a finger-driven vacuum system was incorporated into the system, which achieved microfluidic pumping without external pumps, tubes or power sources. The developed SERS chip achieved fast screening of EGFR E746-A750 mutation in 35 min, reaching a limit of detection down to 100 fM in blood. Due to the simplicity, fast analysis, low cost and quantitative readout, the chip is expected to expand the availability of molecular diagnosis into resource-limited and bedside settings.

ABO Incompatibility Haploidentical Peripheral Blood Stem Cell Transplantation Combined with a Single Cord Blood Unit for Severe Aplastic Anemia Patients

Background: For young severe aplastic anemia (SAA) patients without HLA matched donors, haploidentical hematopoietic stem cell transplantation can be used as an alternative selection. The grafts of classic Beijing protocol in China is a combination of bone marrow and peripheral blood stem cell. For patients with major or minor blood type incompatibility, bone marrow stem cells need to undergo red blood cell or plasma removal treatment. There was rare reports about haploidentical peripheral blood stem cell combined with a single unrelated cord blood unit for ABO incompatibility haploidentical stem cell transplantation in SAA patients. Objective: To observe the efficacy of ABO incompatibility haploidentcial peripheral blood hematopoietic stem cell transplantation combined with a single unrelated cord blood unit for SAA patients. Method s : Fifty-five SAA patients underwent stem cell transplantation from August 2018 to February 2022 in the First Affiliated Hospital of Xi'an Jiaotong Univeristy were divided into two groups based on donor-recipient ABO compatibility, the donor-recipient ABO matched group（BM+PB group）using haploidentical bone marrow and peripheral blood stem cell as grafts, and donor-recipient ABO mismatched group（CB+PB group）, using unrelated cord blood and haploidentical peripheral blood stem cell as grafts. The differences of hematopoietic reconstitution, acute and chronic GVHD, CMV and EB virus infection, PFS, and OS between the two groups were compared. Result s : There were 30 cases in CB+PB group and 27 cases in the BM+PB group. Except for differences in donor-recipient ABO compatibility, the other characteristics of the two groups of patients were comparable((P>0.05). One patient in the BM+PB group had primary graft failure, while the rest of the patients were successfully implanted. There were no significant statistical differences in neutrophil,platelet and red blood cells recovery rates, the incidence of acute and chronic GVHD, CMV, EB virus infection, and PTLD between the two groups of patients(P>0.05). There was no significant statistical difference in OS between the two groups of patients(P>0.05)(Figure 1). Conclusion: For SAA patients with donor-recipient ABO incompatibility could use haploidentical peripheral blood cell combined with unrelated cord blood for transplantation which had compared outcome with haploidentcal bone marrow and peripheral blood cell transplantation.

Use of platelet lysate for in-vitro embryo production and treatment of repeat breeding in cows

Platelet-rich plasma (PRP) is a biological hemocomponent derived from blood after the complete removal of red blood cells and the partial or complete removal of white blood cells to concentrate platelets in an appropriate volume of plasma. Platelets have important growth factors, cytokines, and active metabolites that improve the endometrial environment and positively affect implantation. This study evaluated the effect of the addition of activated PRP (platelets lysate; PL) on in vitro bovine oocyte maturation and embryonic development and the effect of intrauterine (IU) infusion of autologous PL in repeat breeder (RB) cows. Experiment 1 examined the effects of allogeneic PL, fetal calf serum (FCS), mixed PL + FCS, or platelet-poor plasma (PPP) supplementations to in vitro maturation and development media on in vitro oocyte maturation and embryo development in good- and poor-quality oocytes of Japanese Black cows. Experiment 2 examined the IU infusion of autologous PL, 24 h post-insemination, in 21 RB Holstein-Friesian dairy cows. The cleavage rate of good-quality oocytes was higher in the PL group (85.93 ± 2.50%) than in the PPP group (67.16 ± 3.41%) (P < 0.05), while the cleavage rate of the poor-quality oocytes was higher in the PL alone (76.13 ± 4.04%) and mixed PL + FCS treated (73.59 ± 4.22%) groups than in the PPP group (54.64 ± 2.93%) (P < 0.05). The blastocyst rate of the good-quality oocytes was higher in the PL group (40.97 ± 3.03%) than in the FCS (27.97 ± 3.31%) and PPP (25.33 ± 2.15%) groups (P < 0.05). The blastocyst rate of poor-quality oocytes and the hatching rates of both good and poor-quality oocytes showed no significant differences among all groups. The conception rate in the autologous PL-treated group was 41.67% (5/12), while it was 11.11% (1/9) in the control group. The platelets' count in the pregnant PL-treated cows (n = 5; mean ± SEM, 1.07 ± 0.10 × 109/mL) was higher than in the non-pregnant ones (n = 7; 0.67 ± 0.10 × 109/mL) (P < 0.05). In conclusion, allogeneic PL was effective in stimulating the in vitro oocyte maturation and embryonic development in both good and poor-quality bovine oocytes, and post-insemination IU infusion of autologous PL derived from high platelets’ count-PRP would be recommended for the treatment of RB cows.

Establishment of a cascaded microfluidic single cell analysis system for molecular and functional heterogeneity analysis of circulating tumor cells

Single cell heterogeneity analysis of circulating tumor cells (CTCs) is expected to reveal important tumor biological information for the evaluation and prediction of tumor stage and metastatic progression. Although various methods have been proposed to isolate and detect CTCs, consecutive CTCs sorting and in-situ multi-dimensional heterogeneity analysis in a cascaded system remain challenging. Here, a cascaded microfluidic chip was constructed, successively integrating a spiral structure for size-based removal of blood cells and a single cell array structure for the subsequent capture of CTCs, complementing the advantages of high throughput and high separation purity for both structures. Importantly, the molecular and functional heterogeneity analysis of CTCs can be in-situ realized and real-time observed at the single-cell level. Using this system, we captured CTCs and situ-analyzed the heterogeneity of single CTCs from different types of cancer patients, finding that mesenchymal phenotype is present in more CTCs at the advanced stage, and CTCs show active motility and division ability in metastatic patients. Therefore, the cascaded microfluidic chip offers an effective strategy for studying the molecular and functional heterogeneity of single CTCs, providing critical information for the evaluation of tumor progression and metastasis.

Concepts for millimeter wave-based detection of African trypanosomes in field-compatible liquid systems

Abstract Human African Trypanosomiasis (HAT) is caused by the African trypanosome, a single-cell parasite that proliferates in the blood and cerebrospinal fluid of infected patients. Diagnostic measures for this pathogen are currently not sufficiently robust and reliable enough to permit effective disease control procedures. As a consequence, we suggested the development of a new sensor type, combining the selectivity of parasite-specific nucleic acid aptamers with the sensitivity of resonant electromagnetic devices to capture and detect the disease-causing organism. While we accomplished the detection of parasite cells in dehydrated specimens, here we summarize our recent progress toward electromagnetic sensors capable of uncovering parasites in liquid patient samples. We present a technique for the removal of blood cells from blood specimens and the deposition of trypanosome cells on glass microfiber membranes for dielectric spectrometry. Liquid suspensions of trypanosomes are characterized to determine the actual dielectric properties of single parasites and lastly, we present two sensor concepts optimized for the detection in liquids, along with a fabrication technique for the integration of microfluidic sample confinements.

Fast, Reusable, Cell Uniformly Distributed Membrane Filtration Device for Separation of Circulating Tumor Cells.

Isolation of circulating tumor cells (CTCs) is of great significance for the diagnosis, prognosis, and treatment of metastatic cancer. Among CTC capture methods independent of antibodies, membrane filtration-based methods have the advantages of simplicity, rapidity, and high throughput but usually have problems such as clogging, high pressure drop, and impaired cell viability. In this study, we designed and tested a reusable device that used horizontal rotor and fluid-assisted separation to capture CTCs by centrifugal membrane filtration, achieving simple, fast, highly efficient, and viable cell capture on traditional centrifuge. The average capture efficiency was 95.8% for different types of cancer cells with >90% survival, and the removal of white blood cells can reach 99.72% under four times cleaning of the membrane after filtration. A further clinic demo was performed using the device to detect residual leukemic cells in patients; the results showed a 10-fold enrichment of the leukemic cells in peripheral blood samples. Taken together, the simple, robust, and efficient CTC capture device may have the potential for clinic routine detection and analysis of circulating tumor cells.

Enhanced Blood Plasma Extraction Utilising Viscoelastic Effects in a Serpentine Microchannel.

Plasma extraction from blood is essential for diagnosis of many diseases. The critical process of plasma extraction requires removal of blood cells from whole blood. Fluid viscoelasticity promotes cell migration towards the central axis of flow due to differences in normal stress and physical properties of cells. We investigated the effects of altering fluid viscoelasticity on blood plasma extraction in a serpentine microchannel. Poly (ethylene oxide) (PEO) was dissolved into blood to increase its viscoelasticity. The influences of PEO concentration, blood dilution, and flow rate on the performance of cell focusing were examined. We found that focusing performance can be significantly enhanced by adding PEO into blood. The optimal PEO concentration ranged from 100 to 200 ppm with respect to effective blood cell focusing. An optimal flow rate from 1 to 15 µL/min was determined, at least for our experimental setup. Given less than 1% haemolysis was detected at the outlets in all experimental combinations, the proposed microfluidic methodology appears suitable for applications sensitive to haemocompatibility.

RNAseq of Infected Erythrocyte Surface Antigen-Encoding Genes.

Massive parallel sequencing technology has greatly increased the breadth and depth of transcriptomic data that can be captured from P. falciparum samples. This has revolutionized in vitro studies but uptake has been slower in the analysis of clinical samples. The principal barriers are the removal of contaminating white blood cells in a malaria endemic setting and preservation of the RNA. We provide here detailed methods for the collection of purified infected erythrocytes and the preservation and extraction of RNA. We also provide methods for assessing and addressing contaminating RNA from erythroid cells, and a protocol for RNAseq library preparation optimized to maximize yield from low amounts of parasite mRNA. Finally, we provide some examples of RNAseq library characteristics that may fail quality control for other species but are in fact satisfactory for P. falciparum RNAseq.

ELEVATED CIRCULATING ENDOTHELIAL CELLS AND SUCCESS IN ENDOTHELIAL COLONY-FORMING CELLS ISOLATION

Circulating endothelial cells (CEC) have been associated with vascular injury and are described as potential biomarkers for cardiovascular disease (1). Besides, current optimized methodologies enable the isolation of a well-characterized subtype of endothelial progenitor known as Endothelial colony-forming cells (ECFCs) (2,3). Although ECFC isolation methodologies are well described; some discrepancies remain in relation to their isolation efficiency. To evaluate the isolation efficacy ECFCs and CEC frequency in human peripheral blood. The Ethics Research Committee of the University of Campinas approved the experimental procedures and all donors signed the informed consent form. CEC enumeration was assessed by flow cytometry in the peripheral blood of 18 donors. 100 μL of blood (with a leukocyte concentration between 5 and 10 ×10³/μL) was incubated with monoclonal anti-human antibodies (CD45 PerCp, CD133 APC, CD31 FITC, and CD146 PE). FACS lysing solution was used for red blood cell removal. 300,000 events or the total volume of the tube were acquired using a FACSCalibur® flow cytometer (4). To determine the number of cells/μL, the percentage of events was multiplied per number of leucocytes and divided per 100. Cell culture from venous blood was used to isolate ECFCs which were confirmed by their cobblestone morphology and immunophenotyping for endothelial markers (CD31, CD146, CD309, and CD144). ECFC isolation was successful in 9 out of 18 donors (1 male, 8 females; mean age: 35.8 ±11.1 years), and not successful in the other half of donors (2 male, 7 females; mean age: 37.7 ±9.7 years). CEC frequency was significantly higher (p=0.004) in the group with successful ECFC isolation when compared to the group that did not yield ECFCs (2.9 ±1.2 CECs/μL against 1.2 ±0,8 CECs/μL). This suggests that the success in ECFC isolation might be related to the number of CECs, and therefore ECFC mobilization into circulation may also be linked to vascular injury. Increased number of CECs might be associated with the success of ECFCs isolation.

The Multiple Facets of Iron Recycling.

The production of around 2.5 million red blood cells (RBCs) per second in erythropoiesis is one of the most intense activities in the body. It continuously consumes large amounts of iron, approximately 80% of which is recycled from aged erythrocytes. Therefore, similar to the “making”, the “breaking” of red blood cells is also very rapid and represents one of the key processes in mammalian physiology. Under steady-state conditions, this important task is accomplished by specialized macrophages, mostly liver Kupffer cells (KCs) and splenic red pulp macrophages (RPMs). It relies to a large extent on the engulfment of red blood cells via so-called erythrophagocytosis. Surprisingly, we still understand little about the mechanistic details of the removal and processing of red blood cells by these specialized macrophages. We have only started to uncover the signaling pathways that imprint their identity, control their functions and enable their plasticity. Recent findings also identify other myeloid cell types capable of red blood cell removal and establish reciprocal cross-talk between the intensity of erythrophagocytosis and other cellular activities. Here, we aimed to review the multiple and emerging facets of iron recycling to illustrate how this exciting field of study is currently expanding.

Fully Automated, Sample-to-Answer Leukocyte Functional Assessment Platform for Continuous Sepsis Monitoring via Microliters of Blood.

We report a fully automated, sample-to-answer, and label-free leukocyte activation analysis platform for monitoring immune responses in sepsis, by integrating the multidimensional double spiral (MDDS) and isodielectric separation (IDS) subplatforms. The integrated platform can provide rapid and fully automated identification of clinically diagnosed sepsis patients from only 50 μL of peripheral blood volume within 25 min. Many critical innovations were implemented in direct interconnection between the two subplatforms, such as intermediate sample storage and sample transfer, addressing flow rate mismatch (from mL/min to μL/min), and integration of a ridge array for upstream cell focusing in the IDS subplatform. The ridge array in the IDS subplatform can prevent the distortion of electrical profiling due to the residual red blood cells even after the MDDS process. We showed that the integrated platform can separate leukocytes (up to >99.9% red blood cell removal) in the MDDS subplatform and automatically transfer them to the downstream ridge-integrated IDS subplatform for their activation analysis without any apparent ex vivo cell activation and any human intervention. We also demonstrated that the integrated platform can identify differences between leukocytes from human sepsis and healthy subjects significantly (p = 0.0024, 95% confidence interval) by looking into differences in the intrinsic electrical properties of leukocytes. The integrated platform could enable monitoring of host leukocyte function daily or even hourly as a bedside assessment tool, which is currently a critical yet unmet need for managing many critical care patients.

Enhanced Diamagnetic Repulsion of Blood Cells Enables Versatile Plasma Separation for Biomarker Analysis in Blood

A hemolysis-free and highly efficient plasma separation platform enabled by enhanced diamagnetic repulsion of blood cells in undiluted whole blood is reported. Complete removal of blood cells from blood plasma is achieved by supplementing blood with superparamagnetic iron oxide nanoparticles (SPIONs), which turns the blood plasma into a paramagnetic condition, and thus, all blood cells are repelled by magnets. The blood plasma is successfully collected from 4mL of blood at flow rates up to 100µL min-1 without losing plasma proteins, platelets, or exosomes with 83.3±1.64%of plasma volume recovery, which is superior over the conventional microfluidic methods. The theoretical model elucidates the diamagnetic repulsion of blood cells considering hematocrit-dependent viscosity, which allows to determine a range of optimal flow rates to harvest platelet-rich plasma and platelet-free plasma. For clinical validations, it is demonstrated that the method enables the greater recovery of bacterial DNA from the infected blood than centrifugation and the immunoassay in whole blood without prior plasma separation.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Development of a Leukocyte Removal Filter for Hepatitis C Therapy and the Possibility of Multipurpose Treatment

Hepatitis C virus infects and causes inflammation in extrahepatic organs and the liver. Patients with chronic hepatitis C disease receive interferon-based treatment, and both interferon and interferon-free therapies are expensive. We attempted to eliminate hepatitis C virus-infected cells using extracorporeal circulation with a unique filter. We created two types of the filters with syndiotactic polystyrene (SPS) resin sheets in a solution that dissolved hydroxypropyl cellulose (HPC) and cellulose acetate (CA) in oleyl alcohol, respectively. First, hepatitis C virus-positive cells were confirmed in the blood and mononuclear cells of hepatitis C virus-infected volunteers, showing that mononuclear cells phagocytosed the hepatitis C virus. Next, we extracted blood from three healthy volunteers and examined the blood cell removal rate using various filters. The HPC-SPS (fiber diameter, 3.5 μm) showed the highest mononuclear cell removal rate and the lowest platelet removal rate. HPC-SPS reduced hepatitis C virus-positive cells in patients with the hepatitis C virus, and the removal rate of monocytes containing the hepatitis C virus was 77.2%. Thus, HPC-SPS may serve as filter to remove mononuclear cells and granulocytes from blood. Although a technique using extracorporeal circulation is required to apply HPC-SPS, the technical cost is lower than IFN-free drug therapy. We believe that HPC-SPS can be utilized for the treatment of HCV- and leukocyte-related diseases.

Purification of Plasmodium and Babesia- infected erythrocytes using a non-woven fabric filter.

The purification of parasite-infected erythrocytes from whole blood containing leucocytes is crucial for many downstream genetic and molecular assays in parasitology. Current methodologies to achieve this are often costly and time consuming. Here, we demonstrate the successful application of a cheap and simple Non-Woven Fabric (NWF) filter for the purification of parasitized red blood cells from whole blood. NWF filtration was applied to the malaria-parasitized blood of three strains of mice, and one strain of rat, and to Babesia gibsoni parasitized dog blood. Before and after filtration, the white blood cell (WBC) removal rates and red blood cell (RBC) recovery rates were measured. After NWF filter treatment of rodent malaria-infected blood, the WBC removal rates and RBC recovery rates were, for Kunming mice: 99.51%±0.30% and 86.12%±8.37%; for BALB/C mice: 99.61%±0.15% and 80.74%±7.11%; for C57 mice: 99.71%±0.12% and 84.87%±3.83%; for Sprague-Dawley rats: 99.93%±0.03% and 83.30%±2.96%. Microscopy showed WBCs were efficiently removed from infected dog blood samples, and there was no obvious morphological change of B. gibsoni parasites. NWF filters efficiently remove leukocytes from malaria parasite-infected mouse and rat blood, and are also suitable for filtration of B. gibsoni-infected dog blood.

Detection of Melanoma Cells in Whole Blood Samples Using Spectral Imaging and Optical Clearing

Most cancer deaths are associated with metastases resulting from the spread of circulating tumor cells (CTCs) from the primary tumor to vital organs. The existing methods for detection of CTCs as markers of metastasis progression are time consuming with several steps of sample processing, including red blood cell removal, labeling, immunomagnetic capture and isolation, which can lead to loss of CTCs. Here we introduce a method for detection and identification of CTCs using spectral absorption imaging of melanoma cells and optical clearing of whole blood samples. Verification of this approach was performed using phantoms of human melanoma cells and suspensions of mouse melanoma cells of line B16F10 alone and in mixture with blood. A method for improving detection sensitivity has been demonstrated applying optical clearing of mouse blood using biocompatible chemical agents. The findings suggest that the proposed diagnostic platform has the potential to detect quickly CTCs in whole blood samples from patients with melanoma.

High‐Throughput Separation of Microvesicles from Whole Blood Components Using Viscoelastic Fluid

AbstractCell‐derived microvesicles (MVs, also known as microparticles, MPs) are known as important biomarkers of various diseases including cardiovascular diseases, infectious diseases, and cancer. Development of techniques for blood cell removal and MV isolation is required for downstream analyses in biological and clinical applications. A sheathless, label‐free, viscoelastic microfluidic device is proposed for the separation of submicrometer platelet‐derived MPs (PDMPs) from whole blood. Flow rate and channel length dependent performance of the device is evaluated using 2‐µm particles as analogs to platelets and hematocrit (Hct) dependent flow characteristics of red blood cells (RBCs) are observed. In addition, the removal rate of nanoparticles of sizes 300, 500, 700, and 1000 nm is evaluated, and finally, the device is applied to examine the recovery of PDMPs from diluted whole blood sample. At Q = 200 µL min−1 in a 15‐mm long microchannel, RBCs at 5% Hct and platelets are removed to the central outlet without platelet activation, while PDMPs are successfully isolated to the side outlets within ≈2 min. The PDMPs collected from the device are analyzed by flow cytometry. The device exhibits ≈4.8‐fold enhanced isolation performance compared to the centrifugation method.

Proposal of micro plasma extraction device by autonomous trigger control

AbstractIn this paper, we report on the development of a microfluidic device, which extracts plasma from low‐volume whole blood (10 μL), and is aimed at integration with microfluidic system for micro analytical systems. The device autonomously separates blood cells and transfers the separated plasma by steady rotation. Based on the original autonomous fluidic control theory, we developed the principle of automatic timed injection of pressurizing medium into the separation chamber. This realizes the transferring of separated plasma from blood separation chamber to the other place, after separating blood cells by centrifugal force. Through the demonstration on a polydimethylsiloxane microfluidic device, we confirmed that the automatic plasma extraction was successfully implemented as designed. The efficiency of separation was evaluated on the basis of the concentrations of blood cells in samples before and after extraction, and the rate of blood cell removal was 98.5%.

Enabling clinical trials in an academic GMP setting through use of closed, semi-automated manufacturing of allogeneic amniotic epithelial cells

Background & Aim One of the major considerations with regards to the design, usage and ultimately, viability of academic GMP facilities for clinical trials is cost effectiveness. This is particularly the case when clinical trials are supported by scant research funds. In the case of the Monash Health Translation Precinct's Cell Therapy Platform, the Biospherix Xvivo isolator was determined to be a cost-effective method to rapidly create a Class A environment within a Class D room to support manual, open processing. This approach was sufficient for the needs of a small Phase 1, single low dose study where allogeneic amniotic epithelial cells (hAEC) were isolated, cryopreserved and formulated extremely premature babies with chronic lung disease (n=6). For that trial, only 5 placentas were processed. Since the successful completion of this first-in-human trial, the Monash Health Translation Precinct has seen an explosion of hAEC clinical trials including dose-escalation studies in acute stroke, liver cirrhosis, and Crohn's disease. The projected demand on our academic GMP facility is estimated at over 250 processes per year for hAEC isolation alone. Given the labour-intensive nature of hAEC isolation (approx. 4 hours per isolation), and competing demands on the isolator at our academic GMP facility, there is a clear need to move towards closed, automated processing. Methods, Results & Conclusion Accordingly, we applied the use of the RoteaTM (Scinogy Pty Ltd), a benchtop counterflow centrifugal device and single-use kit, to the post-trypsinisation steps of the process. Once separated from the underlying chorion, the amniotic membrane is trimmed into 4 × 4cm pieces and exposed to 0.25% Trypsin-EDTA for 1 hour at 37°C. This is then quenched with 2% (v/v) human serum albumin in saline. The subsequent steps involving red blood cell removal and repeated washing steps (∼ 90 mins in the isolator), were replaced by the use of the RoteaTM (∼10 mins). Furthermore, the RoteaTM steps could be performed outside of the isolator, in a Class D environment with no significant loss in total live cells recovered. This is critical, as it allows staff to clean the isolator and prepare for the entry of incoming material, thus allowing for a level of scalability and flexibility in our current academic GMP manufacturing workflow that could never previously be achieved. Comparing isolation processes alone, this semi-automation will approximately triple the efficiency of our facility due to its enclosed, single-use system.