This work was financially supported by the National Science Foundation for Distinguished Young Scholars (82125018).

Closed System Production of Adherent Cells Based on Dissolvable Microcarriers

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Y.D. is a scientific advisor, and H.X., Y.Z., L.G., M.L., Y.Y., W.L., and X.Y. are employees of Beijing CytoNiche Biotechnology Co. Ltd. The other authors have no commercial, proprietary, or financial interest in the products or companies described in this article. All other authors declare that they have no competing interests.

Both immunotherapy and stem cell therapy utilize live cells as drugs; however, their final products should not be purified or sterilized in the same way as small molecules or viruses. Therefore, the principle of Quality by Design (QbD) should always be kept in mind and practically applied to the Chemical Manufacturing and Control (CMC) process during cell production23. A fully closed cell culture system, as well as a processing system and a filling system, are preferentially considered to meet the...

The CGT industry has witnessed an exponential expansion over the past two decades. The evolution of next-generation medicines is anticipated to treat and cure numerous refractory diseases1. Since the first Food and Drug Administration (FDA) approval of a CGT product, Kymriah, in 2017, CGT-related research and development in the world has continued to grow at a fast rate, with the FDA seeing active investigational new drug applications for CGT increased to 500 in 20182. It had been predicted that the number of approvals of CGT products will likely be 54-74 in the United States by 20302.
While the rapid growth in CGT research and innovation is exciting, there is still a large technological gap between lab research and industrial-scale manufacturing that could deliver these promising medicines to reach as many patients as needed at affordable costs. The current processes adopted for these clinical trials were established in labs for small-scale experiments, and significant efforts are needed to improve and innovate on CGT manufacturing3. There are many types of CGT products, most of them based on live cells, which can be allogenic, autologous, engineered, or natural. These living drugs are much more complex than small molecular entities or biologics, hence making large-scale manufacturing a significant challenge4,5,6. In this work, we demonstrate a large-scale cell production protocol for three anchorage-dependent cells that are widely applied in CGTs. These include human mesenchymal stem/stromal cells (hMSCs), which have been used for cell-based therapy, and HEK293T cells and Vero cells, both of which are used to produce viruses for the genetic engineering of the final therapeutic cell product. Anchorage-dependent cells are commonly cultured on planar systems, which require manual processing. However, manual culture methods require a significant amount of labor and are prone to contamination, which can compromise the quality of the end product. Furthermore, there is no in-line process control, leading to substantial variability in quality between batches7. Taking stem cell therapy as an example, with a promising pipeline of over 200 stem-cell therapy candidates, it is estimated that 300 trillion hMSCs would be needed per year to meet the demands of clinical applications8. Hence, the large-scale manufacturing of therapeutic cells has become a prerequisite to perform these therapeutic interventions with such a high cell demand9.
To preclude the setbacks of planar systems, efforts have been made in developing large-scale manufacturing processes in stirred-tank bioreactors with conventional non-dissolvable microcarriers10,11,12,13, but these suffer from complicated preparation procedures and low cell-harvesting efficiency14. Recently, we have innovated a dissolvable microcarrier for stem cell expansion, aiming to circumvent the challenges of cell harvesting from conventional non-dissolvable commercial microcarriers15. This novel, commercially available GMP-grade 3D dissolvable porous microcarrier, 3D TableTrix, has shown great potential for large-scale cell production. Indeed, 3D culture based on these porous microcarriers could potentially recreate favorable biomimetic microenvironments to promote cell adhesion, proliferation, migration, and activation16. The porous structures and interconnected pore networks of microcarriers could create a larger cell adhesion area and promote the exchange of oxygen, nutrients, and metabolites, thus creating an optimal substrate for in vitro cell expansion17. The high porosity of these GMP-grade 3D dissolvable porous microcarriers enables large-scale expansion of hMSCs, and the ability for the cells to be fully dissolved allows for the efficient harvesting of these expanded cells18. It is also a GMP-grade product and has been registered as a pharmaceutical excipient with the Chinese Center for Drug Evaluation (filing numbers: F20210000003 and F20200000496)19 and the FDA of the United States (FDA, USA; Drug Master File number: 35481)20.
Here, we illustrate an automated closed industrial scale cell production (ACISCP) system18 using these dispersible and dissolvable porous microcarriers for hMSC, HEK293T cell, and Vero cell expansion. We achieved a successful two-tiered expansion of hMSCs (128 cumulative fold expansion in 9 days) from a 5 L bioreactor to a 15 L bioreactor and finally obtained up to 1.1 x 1010 hMSCs from a single batch of production. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Furthermore, we assessed the quality of hMSC products to confirm compliance. We also demonstrated the application of these dissolvable microcarriers for the scaled-up production of two other types of anchorage cells, HEK293T cells and Vero cells, that are extensively applied in the CGT industry. The peak cell density of HEK293T cells reached 1.68 x 107 cells/mL, whereas the peak density of Vero cells reached 1.08 x 107 cells/mL. The ACISCP system could be adapted to culture a variety of adherent cells, and it could potentially become a powerful platform contributing to expediting the industrialization of CGT.

<table><tbody><tr><td>0.25% trypsin EDTA</td><td>BasalMedia</td><td>S310JV</td><td>Used for 2D cell harvest.</td></tr><tr><td>3D FloTrix Digest</td><td>CytoNiche Biotech</td><td>R001-500</td><td>This is a reagent that specifically dissolves 3D TableTrix microcarriers.</td></tr><tr><td>3D FloTrix MSC Serum Free Medium</td><td>CytoNiche Biotech</td><td>RMZ112</td><td>This is a serum-free,animal-free medium for mesenchymal stem cell expansion and maintenance in 2D planar culture as well as 3D culture on 3D TableTrix microcarriers.</td></tr><tr><td>3D FloTrix Single-Use Filtration Module</td><td>CytoNiche Biotech</td><td>R020-00-10</td><td>This module contains 0.22 &amp;mu;m capsule filters used for filtration of culture medium and digest solution.</td></tr><tr><td>3D FloTrix Single-Use Storage Bag (10 L)</td><td>CytoNiche Biotech</td><td>R020-00-03</td><td>Used as feed bag for 5 L bioreactor.</td></tr><tr><td>3D FloTrix Single-Use Storage Bag (3 L)</td><td>CytoNiche Biotech</td><td>R020-00-01</td><td>Used as cell seeding or transfer bags.</td></tr><tr><td>3D FloTrix Single-Use Storage Bag (50 L)</td><td>CytoNiche Biotech</td><td>R020-00-04</td><td>Used as feed bag for 15 L bioreactor.</td></tr><tr><td>3D FloTrix vivaPACK Disposable Fill&amp;amp;Finish Consumable Kit</td><td>CytoNiche Biotech</td><td>PACK-01-01</td><td>This is a standard kit adapted to 3D vivaPACK fill and finish system.</td></tr><tr><td>3D FloTrix vivaPACK fill and finish system for cells</td><td>CytoNiche Biotech</td><td>vivaPACK</td><td>This system is a closed liquid handling device, with automated mixing and gas exhausting functions. Cells resuspended in cryopreservation buffer can be rapidly and evenly aliquoted into 20 bags per batch.</td></tr><tr><td>3D FloTrix vivaPREP PLUS cell processing system</td><td>CytoNiche Biotech</td><td>vivaPREP PLUS</td><td>This system is a continuous flow centrifuge-based device.Cells can be concentrated, washed, and resuspended under completely closed procedures.</td></tr><tr><td>3D FloTrix vivaPREP PLUS Disposable Cell Processing Kit</td><td>CytoNiche Biotech</td><td>PREP-PLUS-00</td><td>This is a standard kit adapted to 3D vivaPREP PLUS cell processing.</td></tr><tr><td>3D FloTrix vivaSPIN&amp;nbsp; bioreactor 15 L</td><td>CytoNiche Biotech</td><td>FTVS15</td><td>This bioreactor product employs a controller, a 15 L glass stirred-tank vessel, and assessories. A special perfusion tube is available.</td></tr><tr><td>3D FloTrix vivaSPIN&amp;nbsp; bioreactor 5 L</td><td>CytoNiche Biotech</td><td>FTVS05</td><td>This bioreactor product employs a controller, a 5 L glass stirred-tank vessel, and assessories.A special perfusion tube is available.</td></tr><tr><td>3D FloTrix vivaSPIN Closed System Consumable Pack (10/15 L)</td><td>CytoNiche Biotech</td><td>R020-10-10</td><td>This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 15 L, containing sampling bags.</td></tr><tr><td>3D FloTrix vivaSPIN Closed System Consumable Pack (2/5 L)</td><td>CytoNiche Biotech</td><td>R020-05-10</td><td>This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 5 L, containing sampling bags.</td></tr><tr><td>3D TableTrix microcarriers G02</td><td>CytoNiche Biotech</td><td>G02-10-10g</td><td>These porous and degradable microcarriers are suitable for HEK293T cell culture. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.</td></tr><tr><td>3D TableTrix microcarriers V01</td><td>CytoNiche Biotech</td><td>V01-100-10g</td><td>These porous and degradable microcarriers are suitable for adherent cell culture, they come as non-sterilized microcarriers that need to be autoclaved in PBS before use. They are especially suitable for vaccine production.</td></tr><tr><td>3D TableTrix microcarriers W01</td><td>CytoNiche Biotech</td><td>W01-10-10g (single-use packaging);&lt;br/&gt; W01-200 (tablets)</td><td>These porous and degradable microcarriers are suitable for adherent cell culture, especially for cells that need to be harvested as end products. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.The product has obtained 2 qualifications for pharmaceutical excipients from CDE, with the registration numbers of [F20200000496; F20210000003]. It has also received DMF qualification for pharmaceutical excipients from FDA, with the registration number of [DMF:35481]</td></tr><tr><td>APC anti-human CD45 Antibody</td><td>BioLegend</td><td>368512</td><td>Used in flow cytometry for MSC identity assessment</td></tr><tr><td>Calcein-AM/PI Double Staining Kit</td><td>Dojindo</td><td>C542</td><td>Calcein-AM/PI Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stain viable and dead cells, respectively.</td></tr><tr><td>Cap for EZ Top Container Closures for NALGENE-containers (500mL)</td><td>Saint-Gobain</td><td>CAP-38</td><td>Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.</td></tr><tr><td>C-Flex Tubing, Formulation 374 (0.25 in x 0.44 in)</td><td>Saint-Gobain</td><td>374-250-3</td><td>Used for tube welding and disconnection.</td></tr><tr><td>CryoMACS Freezing Bag 50</td><td>Miltenyi Biotec&amp;nbsp;</td><td>200-074-400</td><td>Used for expanding the 3D FloTrix vivaPACK Disposable Fill&amp;amp;Finish Consumable Kit.</td></tr><tr><td>Dimethyl Sulfate (DMSO)&amp;nbsp;</td><td>Sigma</td><td>D2650-100mL</td><td>Used for preparation of cryopreservation solution.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>BasalMedia</td><td>L120KJ</td><td>Used for cultivation of HEK293T and Vero cells.</td></tr><tr><td>DURAN Original GL 45 Laboratory bottle (2 L)</td><td>DWK life sciences</td><td>218016357</td><td>Used for waste collection from the 5 L bioreactor.</td></tr><tr><td>DURAN Original GL 45 Laboratory bottle (5 L)</td><td>DWK life sciences</td><td>218017353</td><td>Used for waste collection from the 15 L bioreactor.</td></tr><tr><td>DURAN Original GL 45 Laboratory bottle (500 mL)</td><td>DWK life sciences</td><td>218014459</td><td>Used for supplementary bottle of 0.1 M NaOH.</td></tr><tr><td>EZ Top Container Closures for NALGENE-containers (500mL)</td><td>Saint-Gobain</td><td>EZ500 ML-38-2</td><td>Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.</td></tr><tr><td>Fetal bovine serum (FBS) superior quality</td><td>Wisent</td><td>086-150</td><td>Used for cultivation of HEK293T cells.</td></tr><tr><td>FITC anti-human CD14 Antibody</td><td>BioLegend</td><td>301804</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>FITC anti-human CD34 Antibody</td><td>BioLegend</td><td>343504</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>FITC anti-human CD90 (Thy1) Antibody</td><td>BioLegend</td><td>328108</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>Flow cytometry</td><td>Beckman Coulter</td><td>CytoFLEX</td><td>Used for cell identity assessment.</td></tr><tr><td>Fluorescence Cell Analyzer</td><td>Alit life science</td><td>Countstar Rigel S2</td><td>Used for cell counting. Cell viability can be calculated by staining with AO/PI dyes.</td></tr><tr><td>GL 45 Multiport Connector Screw Cap with 2 ports&amp;nbsp;</td><td>DWK life sciences</td><td>292632806</td><td>Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.</td></tr><tr><td>Glucose Meter</td><td>Sinocare</td><td>6243578</td><td>Used for detecting glucose concentration in cell culture medium and supernatant.</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), with calcium and magnesium</td><td>Gibco</td><td>14025092</td><td>Used for preparation of digest solution.</td></tr><tr><td>Human Albumin 20% Behring (HSA)</td><td>CSL Behring</td><td>N/A</td><td>Used for preparation of wash buffer.</td></tr><tr><td>Inverted fluorescent microscope</td><td>OLYMBUS</td><td>CKX53SF</td><td>Used for brifgt field and fluorescent observation and imaging.</td></tr><tr><td>Nalgene Measuring Cylinder (500 mL)</td><td>Thermo Scientific</td><td>3662-0500PK</td><td>Used for calibrating the liquid handling volume speed of peristaltic pumps.</td></tr><tr><td>Newborn calf serum (NBS) superfine</td><td>MINHAI BIO</td><td>SC101.02</td><td>Used for cultivation of Vero cells.</td></tr><tr><td>OriCell human mesenchymal stem cell adipogenic differentiation and characterization kit</td><td>Cyagen</td><td>HUXUC-90031</td><td>Used for tri-lineage differentiation of hUCMSCs.</td></tr><tr><td>OriCell human mesenchymal stem cell chondrogenic differentiation and characterization kit</td><td>Cyagen</td><td>HUXUC-90041</td><td>Used for tri-lineage differentiation of hUCMSCs.</td></tr><tr><td>OriCell human mesenchymal stem cell osteogenic differentiation and characterization kit</td><td>Cyagen</td><td>HUXUC-90021</td><td>Used for tri-lineage differentiation of hUCMSCs.</td></tr><tr><td>PE anti-human CD105 Antibody</td><td>BioLegend</td><td>800504</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>PE anti-human CD19 Antibody</td><td>BioLegend</td><td>302208</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>PE anti-human CD73 (Ecto-5&#039;-nucleotidase) Antibody</td><td>BioLegend</td><td>344004</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>PE anti-human HLA-DR Antibody</td><td>BioLegend</td><td>307605</td><td>Used in flow cytometry for MSC identity assessment.</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Wisent</td><td>311-010-CL</td><td>Used in autoclaving of glass vessel and V01 microcarriers, and replacement of culture medium.</td></tr><tr><td>Sani-Tech Platinum Cured Sanitary Silicone Tubing (0.13 in x 0.25 in)</td><td>Saint-Gobain</td><td>ULTRA-C-125-2F</td><td>Used for solution transfering driven by peristaltic pumps.</td></tr><tr><td>Sterile Saline</td><td>Hopebiol</td><td>HBPP008-500</td><td>Used for preparation of wash buffer.</td></tr><tr><td>Trypzyme Recombinant Trypsin</td><td>BasalMedia</td><td>S342JV</td><td>This reagent is used for bead-to-bead transfer of HEK293T and Vero cells.</td></tr><tr><td>Tube Sealer</td><td>Yingqi Biotech</td><td>Tube Sealer I</td><td>This sealer is compatible with both C-Flex tubing and PVC tubing.</td></tr><tr><td>Tube Welder for PVC tubing</td><td>Chu Biotech</td><td>Tube Welder Micro I</td><td>Used for welding of PVC tubing.</td></tr><tr><td>Tube Welder for TPE tubing</td><td>Yingqi Biotech</td><td>Tube Welder I-V2</td><td>Used for welding of TPE tubing.</td></tr><tr><td>ViaStain AO / PI Viability Stains</td><td>Nexcelom</td><td>CS2-0106-25mL</td><td>Dual-Fluorescence Viability, using acridine orange (AO) and propidium iodide (PI), is the recommended method for accurate viability analysis of primary cells, such as PBMCs, and stem cells in samples containing debris.</td></tr></tbody></table>

large-scale cell production based on gmp-grade dissolvable porous microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

The ACISCP platform is a fully closed system that employs a series of stirred-tank bioreactors for scale-up expansion, a cell processing system for automated cell harvesting and formulation, and a cell filling system (Figure 1). Adherent cells attach to the porous microcarriers, which can be dispersed into the bioreactor, thus achieving the suspended cultivation of adherent cells.
Following the protocol as described, 2.5 x 108 hMSCs with 10 g of W01 mic...

Watch this Scientific Journal Video about Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers at JoVE.com

Author Spotlight: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...

large-scale-cell-production-based-on-gmp-grade-dissolvable-porous

Research

JoVE Journal

Bioengineering

3.1K Views.  Tsinghua University. De cel- en gentherapie-industrie is de afgelopen twee decennia getuige geweest van een exponentiële expansie. Er bestaat echter een grote technologische kloof tussen laboratoriumonderzoek en de productie van industriële vaardigheden. In overeenstemming met het regelgevingsbeleid en om te voldoen aan de groene eisen van de CGT-industrie, zijn geautomatiseerde gesloten systemen ingevoerd om celtherapieën te produceren.Voor aanhangende cellen is kweek met behulp van microcarriers in sc...

Video: Auteur in de schijnwerpers: Bevordering van de productie van celtherapie met oplosbare microcarriers 

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

Developmental Biology

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Biology

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market ...

Biochemistry

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

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