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.
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 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.
The human umbilical cord was obtained from Beijing Tsinghua Changgeng Hospital. All procedures and protocols regarding the acquisition, isolation, and culture of human umbilical cord mesenchymal stem cells (UCMSCs) were conducted with informed consent and with the approval of the Ethics Committee of Beijing Tsinghua Changgeng Hospital (filing number 22035-4-02), and the procedures and protocols complied with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
1. Monolayer culture of hMSCs, HEK293T cells, and Vero cells
2. Preparation of the stirred-tank bioreactor before cell seeding
3. Cell seeding, culture, and harvest in a 5 L stirred-tank bioreactor
4. Cell formulation, fill, and finish
5. Characterization of the cell quality
6. HEK293T expansion on G02 microcarriers in the stirred-tank bioreactor
7. Vero cell expansion on V01 microcarriers in the stirred-tank bioreactor
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 microcarriers were firstly inoculated in a 5 L stirred-tank bioreactor for an initial expansion. Around 2.9 x 109 cells were concentrated and washed by the cell processing system on day 4, out of which 1.0 x 109 harvested cells were passaged to the 15 L stirred-tank bioreactor with 40 g of W01 microcarriers for the second phase of cultivation. Eventually, 1.1 x 1010 MSCs were harvested, formulated, and aliquoted into over 70 formulation bags on day 9 (Figure 2A). The cell number, viability, and glucose consumption were monitored daily (Figure 2B,C). A cumulative 128-fold expansion was achieved; meanwhile, the cell viability was kept higher than 90%. The intake of glucose exhibited a negative correlation with cell expansion, showing metabolism associated with healthy cell growth.
The dissolvable porous microcarriers can be pre-sterilized and come in a single-use closed system packaging, suitable for GMP (Figure 3A). The cells could attach to the surface of the microcarriers and the inner wall of the macropores (Figure 3B). By using a fluorescent dye, such as Calcein-AM/PI Cell Double Staining Kit, the cells inside the porous microspheres could be visualized (Figure 3C). Merging bright-field images with fluorescent images helped to analyze the uniformity of the cell distribution. The live cell staining also showed a strong association with cell counting measured by either manual sampling or the online live cell counting probe.
In addition to the tremendous demand for high cell quantities, critical quality attribute (CQA) assessments and release tests of MSCs are indispensable, as without these, cells could not be sterilized or extensively purified post-culturing. To assure the cell quality, 3D-expanded MSCs can be sampled at each stage of the ACISCP platform. MSCs freshly harvested from the ACISCP platform retained classical phenotypic markers22, with an expression of >95% and <2% for the positive markers (CD73, CD90, CD105) and negative markers (HLA-DR, CD34, CD45, CD19, CD14), respectively (Figure 4A). The cryopreserved cells were thawed and inoculated to a 2D planar flask and showed good adhesion ability, normal expansion behavior (Figure 4B), and a typical spindle shape with spiral growth (Figure 4C). Furthermore, the cells also maintained their capability to differentiate into osteogenic, adipogenic, and chondrogenic lineages (Figure 4D).
Moreover, HEK293T cells and Vero cells were tested in the ACISCP platform as well. For HEK293T cell culture, the inoculum cell concentration was 5 x 105 cells/mL in the 5 L bioreactor. After the bead-to-bead transfer process, the peak cell concentration reached 1.68 x 107 cells/mL in the 15 L bioreactor (Figure 5A), and the HEK293T cells maintained high viability (Figure 5B). For the Vero cell culture, the inoculum cell concentration was 2 x 105 cells/mL in the 5 L bioreactor. After the bead-to-bead transfer process, the peak cell concentration achieved was 1.08 x 107 cells/mL in the 15 L bioreactor (Figure 6A), and the Vero cells maintained high viability as well (Figure 6B).
Figure 1: Schematic roadmap of hMSC production via the ACISCP platform. This figure has been modified from published literature18. Please click here to view a larger version of this figure.
Figure 2: Cell expansion of hMSCs via the ACISCP platform. (A) The ACISCP platform consists of a series of stirred-tank bioreactors, a cell processing system, and a cell filling system. (B) Growth curve of hMSCs in the two-tiered expansion process, (C) and the glucose consumption rate during the process. Please click here to view a larger version of this figure.
Figure 3: Characterization of hMSC growth on the W01 microcarrier. This figure has been modified from published literature18. (A) W01 microcarriers are prepared with single-use closed-system packaging. (B) Scanning electron microscope images of empty microcarriers and microcarriers cultured with adherent hMSCs. The white triangular heads indicate cells on the surface of microcarriers; scale bar = 100 µm. (C) Representative merged bright-field and fluorescence (live cell staining) images of hMSCs cultured on microcarriers from day 1 to day 4; scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 4: Cell identity assessment of hMSCs harvested from the ACISCP platform. (A) The surface markers of hMSCs were characterized by flow cytometry. All the positive markers (>95%) and negative markers (<2%) met the criteria. (B) Cryopreserved 3D-expanded hMSCs exhibited normal expansion behavior after thawing and re-plating to 2D flasks. (C) Morphology of the 3D-expanded hMSCs; scale bar = 50 µm. (D) Tri-lineage differentiation of the 3D-expanded hMSCs. Osteogenic, adipogenic, and chondrogenic capabilities were assessed by Alizarin Red, Oil Red, and Alcian Blue staining, respectively. Please click here to view a larger version of this figure.
Figure 5: Cell expansion of HEK293T cells via the ACISCP platform. (A) HEK293T cell density in the 5 L and 15 L bioreactors. (B) Bright-field (BF) image, Calcein-AM-stained (Live), and PI-stained (Dead) HEK293T cells cultured on G02 microcarriers; scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 6: Cell expansion of Vero cells via the ACISCP platform. (A) Vero cell density in the 5 L and 15 L bioreactors. (B) Bright-field (BF) image, Calcein-AM-stained (Live), and PI-stained (Dead) Vero cells cultured on V01 microcarriers; scale bar = 400 µm. Please click here to view a larger version of this figure.
Table 1: Parameter settings of the stirred-tank bioreactor. Please click here to download this Table.
Table 2: Parameter settings of automated medium exchange for hMSC cultivation. Please click here to download this Table.
Table 3: Parameter settings of the cell processing system for hMSC passage and formulation. Please click here to download this Table.
Table 4: Medium exchange regime and agitation regime for HEK293T and Vero cells. Please click here to download this Table.
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 requirements. In this study, we have presented a protocol for utilizing an ACISCP platform based on GMP-grade, dissolvable, and porous 3D microcarriers to perform a two-tiered expansion of hMSCs. Some researchers have explored the feasibility of using bioreactors together with microcarriers to expand hMSCs in vitro, but the majority of reported studies have shown that an increase in scale would lead to a decrease in hMSC yield, from 6.1 x 105 cells/mL or even 12.5 x 105 cells/mL in 100 mL of working volume to 2.7 x 105 cells/mL in 2 L of working volume24,25,26. Even though stirred-tank bioreactors exhibit the highest scalability, key issues such as the structural characteristics of the vessel, the impeller type, the tip speed, the volumetric mass transfer coefficient, kLa, of oxygen, and the power number, should be thoroughly considered24. Distinct from previous stirred-tank bioreactors, which were usually designed for the cultivation of microbes or domesticated anchorage-independent cells like CHO cells, the bioreactor used in this study was specifically designed for microcarrier-based culture and is, thus, able to satisfy the different process needs of nutrient exchange strategies. In addition to differences in the process of cell cultivation, the ACISCP platform employs a continuous flow centrifuge-based cell processing system together with a cell filling system, in contrast to conventional harvesting procedures for producing large amounts of cells performed by repeated manual work, thus guaranteeing a high handling efficacy. Advanced automated devices have a high yield in one-batch production.
Most stem cells are anchorage-dependent; thus, they demand microcarriers for cell manufacturing. Conventionally, previous commercial microcarriers were formulated to leverage different surface characteristics, thus achieving different cell expansion fold numbers. In previous research, the use of Cytodex type 1 microcarriers for porcine bone marrow MSCs produced a cell density of about 4 x 105 cells/mL, while the use of gelatin-coated Cytodex type 3 produced comparable cell numbers (3.8 x 105 cells/mL) to human placental MSCs27. Nevertheless, Cytodex type 1 and type 3 are non-dissolvable, and, thus, the trypsinization process during cell harvesting impacts not only the cell recovery yield but also the viability and quality. Hence, there are no successful cases of using conventional microcarriers for CGT products, where cells as the final products cannot undergo extensive downstream purification processes to eliminate the contamination of non-dissolvable microcarriers28. By contrast, W01 microcarriers are fully dissolvable, meaning that the cell products can be easily harvested by degradation of the microcarrier. This microcarrier product also comes in fully closed system packaging, meaning an operation process compliant with GMP could be achieved easily. We have shown that passaging with a two-tiered expansion process can be easily achieved with W01 microcarriers, and as high as 1.1 x 1010 total cells can be harvested in a single batch; indeed, this could only be achieved in 50 L bioreactors in previous reports29,30. In this work, the peak cell density inside the stirred-tank bioreactor reached approximately 11.6 x 105 cells/mL before harvest. Thus, it could be expected that one could easily achieve 1 x 1011 cells per batch with a 100-200 L bioreactor in the ACISCP platform, which would mean that thousand batches per year could easily meet the demand for 300 trillion hMSCs each year.
Since cells have varied characteristics, different types of 3D microcarriers are produced and are available for testing the compatibility between a specific cell and a microcarrier. For non-cell end products in CGT, microcarrier-based scale-ups for HEK293T cells and Vero cells in bioreactors have been reported to reach 1.5 x 106 cells/mL and 3.3 x 106 cells/mL, respectively31. We used dissolvable porous microcarriers, G02 and V01, in the ACISCP platform to expand HEK293T cells and Vero cells, respectively, and the peak cell densities of both cells reached above 1 x 107 cells/mL. Furthermore, the degradability of 3D microcarriers can be beneficial for the production of biological products of intracellular viruses, as the cells could be easily separated from the microcarriers. Critically, the ACISCP platform represents an established new biomanufacturing process that meets both the quantity and quality requirements for cell and gene therapy products. We anticipate that our large-scale cell production platform will act as an essential tool to enable the development of cell and gene therapies.
Despite the advantages of the platform discussed above, several constraints remain to be circumvented in the future. Firstly, the bioreactors employed in the ACISCP platform in the current form are still equipped with glass tanks, which require complicated washing procedures and cleaning verification to meet the criteria of GMP. A single-use stirred-tank bioreactor system could be potentially employed in the future to produce an intact set of single-use consumable kits. Secondly, different from domesticated cell lines, hMSCs are primary cell strains isolated from individual donors and varied tissues, which means hMSCs exhibit heterogeneity. Therefore, the protocol described in this paper serves as a reference, while systematic process developments are required to achieve the technical migration of the ACISCP platform. Thirdly, even though our co-workers have preliminarily tested the feasibility of producing the vaccinia virus with Vero cells32, whether good performance for viral production in the 3D expansion of HEK293T cells could be achieved remains to be further validated. In conclusion, the large-scale cell production method of the ACISCP platform, based on dissolvable porous microcarriers and a series of automated closed systems, offers an opportunity to enhance production efficiency and refine quality control at an industrial scale for the manufacturing of CGT products.
The authors have nothing to disclose.
This work was financially supported by the National Science Foundation for Distinguished Young Scholars (82125018).
0.25% trypsin EDTA | BasalMedia | S310JV | Used for 2D cell harvest. |
3D FloTrix Digest | CytoNiche Biotech | R001-500 | This is a reagent that specifically dissolves 3D TableTrix microcarriers. |
3D FloTrix MSC Serum Free Medium | CytoNiche Biotech | RMZ112 | 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. |
3D FloTrix Single-Use Filtration Module | CytoNiche Biotech | R020-00-10 | This module contains 0.22 μm capsule filters used for filtration of culture medium and digest solution. |
3D FloTrix Single-Use Storage Bag (10 L) | CytoNiche Biotech | R020-00-03 | Used as feed bag for 5 L bioreactor. |
3D FloTrix Single-Use Storage Bag (3 L) | CytoNiche Biotech | R020-00-01 | Used as cell seeding or transfer bags. |
3D FloTrix Single-Use Storage Bag (50 L) | CytoNiche Biotech | R020-00-04 | Used as feed bag for 15 L bioreactor. |
3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit | CytoNiche Biotech | PACK-01-01 | This is a standard kit adapted to 3D vivaPACK fill and finish system. |
3D FloTrix vivaPACK fill and finish system for cells | CytoNiche Biotech | vivaPACK | 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. |
3D FloTrix vivaPREP PLUS cell processing system | CytoNiche Biotech | vivaPREP PLUS | This system is a continuous flow centrifuge-based device.Cells can be concentrated, washed, and resuspended under completely closed procedures. |
3D FloTrix vivaPREP PLUS Disposable Cell Processing Kit | CytoNiche Biotech | PREP-PLUS-00 | This is a standard kit adapted to 3D vivaPREP PLUS cell processing. |
3D FloTrix vivaSPIN bioreactor 15 L | CytoNiche Biotech | FTVS15 | This bioreactor product employs a controller, a 15 L glass stirred-tank vessel, and assessories. A special perfusion tube is available. |
3D FloTrix vivaSPIN bioreactor 5 L | CytoNiche Biotech | FTVS05 | This bioreactor product employs a controller, a 5 L glass stirred-tank vessel, and assessories.A special perfusion tube is available. |
3D FloTrix vivaSPIN Closed System Consumable Pack (10/15 L) | CytoNiche Biotech | R020-10-10 | This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 15 L, containing sampling bags. |
3D FloTrix vivaSPIN Closed System Consumable Pack (2/5 L) | CytoNiche Biotech | R020-05-10 | This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 5 L, containing sampling bags. |
3D TableTrix microcarriers G02 | CytoNiche Biotech | G02-10-10g | 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. |
3D TableTrix microcarriers V01 | CytoNiche Biotech | V01-100-10g | 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. |
3D TableTrix microcarriers W01 | CytoNiche Biotech | W01-10-10g (single-use packaging); W01-200 (tablets) |
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] |
APC anti-human CD45 Antibody | BioLegend | 368512 | Used in flow cytometry for MSC identity assessment |
Calcein-AM/PI Double Staining Kit | Dojindo | C542 | 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. |
Cap for EZ Top Container Closures for NALGENE-containers (500mL) | Saint-Gobain | CAP-38 | 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. |
C-Flex Tubing, Formulation 374 (0.25 in x 0.44 in) | Saint-Gobain | 374-250-3 | Used for tube welding and disconnection. |
CryoMACS Freezing Bag 50 | Miltenyi Biotec | 200-074-400 | Used for expanding the 3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit. |
Dimethyl Sulfate (DMSO) | Sigma | D2650-100mL | Used for preparation of cryopreservation solution. |
Dulbecco's Modified Eagle Medium (DMEM) | BasalMedia | L120KJ | Used for cultivation of HEK293T and Vero cells. |
DURAN Original GL 45 Laboratory bottle (2 L) | DWK life sciences | 218016357 | Used for waste collection from the 5 L bioreactor. |
DURAN Original GL 45 Laboratory bottle (5 L) | DWK life sciences | 218017353 | Used for waste collection from the 15 L bioreactor. |
DURAN Original GL 45 Laboratory bottle (500 mL) | DWK life sciences | 218014459 | Used for supplementary bottle of 0.1 M NaOH. |
EZ Top Container Closures for NALGENE-containers (500mL) | Saint-Gobain | EZ500 ML-38-2 | 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. |
Fetal bovine serum (FBS) superior quality | Wisent | 086-150 | Used for cultivation of HEK293T cells. |
FITC anti-human CD14 Antibody | BioLegend | 301804 | Used in flow cytometry for MSC identity assessment. |
FITC anti-human CD34 Antibody | BioLegend | 343504 | Used in flow cytometry for MSC identity assessment. |
FITC anti-human CD90 (Thy1) Antibody | BioLegend | 328108 | Used in flow cytometry for MSC identity assessment. |
Flow cytometry | Beckman Coulter | CytoFLEX | Used for cell identity assessment. |
Fluorescence Cell Analyzer | Alit life science | Countstar Rigel S2 | Used for cell counting. Cell viability can be calculated by staining with AO/PI dyes. |
GL 45 Multiport Connector Screw Cap with 2 ports | DWK life sciences | 292632806 | 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. |
Glucose Meter | Sinocare | 6243578 | Used for detecting glucose concentration in cell culture medium and supernatant. |
Hank's Balanced Salt Solution (HBSS), with calcium and magnesium | Gibco | 14025092 | Used for preparation of digest solution. |
Human Albumin 20% Behring (HSA) | CSL Behring | N/A | Used for preparation of wash buffer. |
Inverted fluorescent microscope | OLYMBUS | CKX53SF | Used for brifgt field and fluorescent observation and imaging. |
Nalgene Measuring Cylinder (500 mL) | Thermo Scientific | 3662-0500PK | Used for calibrating the liquid handling volume speed of peristaltic pumps. |
Newborn calf serum (NBS) superfine | MINHAI BIO | SC101.02 | Used for cultivation of Vero cells. |
OriCell human mesenchymal stem cell adipogenic differentiation and characterization kit | Cyagen | HUXUC-90031 | Used for tri-lineage differentiation of hUCMSCs. |
OriCell human mesenchymal stem cell chondrogenic differentiation and characterization kit | Cyagen | HUXUC-90041 | Used for tri-lineage differentiation of hUCMSCs. |
OriCell human mesenchymal stem cell osteogenic differentiation and characterization kit | Cyagen | HUXUC-90021 | Used for tri-lineage differentiation of hUCMSCs. |
PE anti-human CD105 Antibody | BioLegend | 800504 | Used in flow cytometry for MSC identity assessment. |
PE anti-human CD19 Antibody | BioLegend | 302208 | Used in flow cytometry for MSC identity assessment. |
PE anti-human CD73 (Ecto-5'-nucleotidase) Antibody | BioLegend | 344004 | Used in flow cytometry for MSC identity assessment. |
PE anti-human HLA-DR Antibody | BioLegend | 307605 | Used in flow cytometry for MSC identity assessment. |
Phosphate Buffered Saline (PBS) | Wisent | 311-010-CL | Used in autoclaving of glass vessel and V01 microcarriers, and replacement of culture medium. |
Sani-Tech Platinum Cured Sanitary Silicone Tubing (0.13 in x 0.25 in) | Saint-Gobain | ULTRA-C-125-2F | Used for solution transfering driven by peristaltic pumps. |
Sterile Saline | Hopebiol | HBPP008-500 | Used for preparation of wash buffer. |
Trypzyme Recombinant Trypsin | BasalMedia | S342JV | This reagent is used for bead-to-bead transfer of HEK293T and Vero cells. |
Tube Sealer | Yingqi Biotech | Tube Sealer I | This sealer is compatible with both C-Flex tubing and PVC tubing. |
Tube Welder for PVC tubing | Chu Biotech | Tube Welder Micro I | Used for welding of PVC tubing. |
Tube Welder for TPE tubing | Yingqi Biotech | Tube Welder I-V2 | Used for welding of TPE tubing. |
ViaStain AO / PI Viability Stains | Nexcelom | CS2-0106-25mL | 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. |