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

<ol>
	<li>Golchin, A., Farahany, T. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+products:+Cellular+therapy+and+FDA+approved+products.">Biological products: Cellular therapy and FDA approved products.</a> Stem Cell Reviews and Reports. 15 (2), 166-175 (2019).</li><li>Young, C. M., Quinn, C., Trusheim, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+cell+and+gene+therapy+potential+patient+and+financial+impact:+US+projections+of+product+approvals,+patients+treated,+and+product+revenues.">Durable cell and gene therapy potential patient and financial impact: US projections of product approvals, patients treated, and product revenues.</a> Drug Discovery Today. 27 (1), 17-30 (2022).</li><li>Elverum, K., Whitman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivering+cellular+and+gene+therapies+to+patients:+solutions+for+realizing+the+potential+of+the+next+generation+of+medicine.">Delivering cellular and gene therapies to patients: solutions for realizing the potential of the next generation of medicine.</a> Gene Therapy. 27 (12), 537-544 (2020).</li><li>Blache, U., Popp, G., D&#252;nkel, A., Koehl, U., Fricke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+solutions+for+manufacture+of+CAR+T+cells+in+cancer+immunotherapy.">Potential solutions for manufacture of CAR T cells in cancer immunotherapy.</a> Nature Communications. 13 (1), 5225 (2022).</li><li>Lee, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+process+scale-up+challenges+for+commercial-scale+manufacturing+of+allogeneic+pluripotent+stem+cell+products.">Cell culture process scale-up challenges for commercial-scale manufacturing of allogeneic pluripotent stem cell products.</a> Bioengineering. 9 (3), 92 (2022).</li><li>Emerson, J., Glassey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocess+monitoring+and+control:+Challenges+in+cell+and+gene+therapy.">Bioprocess monitoring and control: Challenges in cell and gene therapy.</a> Current Opinion in Chemical Engineering. 34, 100722 (2021).</li><li>Robb, K. P., Fitzgerald, J. C., Barry, F., Viswanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell+therapy:+progress+in+manufacturing+and+assessments+of+potency.">Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency.</a> Cytotherapy. 21 (3), 289-306 (2019).</li><li>Olsen, T. R., Ng, K. S., Lock, L. T., Ahsan, T., Rowley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peak+MSC-Are+we+there+yet.">Peak MSC-Are we there yet.</a> Frontiers in Medicine. 5, 178 (2018).</li><li>. Roots Analysis. Stem Cell Therapy Contract Manufacturing (CMO) Market, 2019 - 2030 Available from: <a target="_blank" href="https://www.rootsanalysis.com/reports/view_document/stem-cell-therapy-contract-manufacturing-market-2019-2030/271.html">https://www.rootsanalysis.com/reports/view_document/stem-cell-therapy-contract-manufacturing-market-2019-2030/271.html</a> (2019)</li><li>Elseberg, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier-based+expansion+process+for+hMSCs+with+high+vitality+and+undifferentiated+characteristics.">Microcarrier-based expansion process for hMSCs with high vitality and undifferentiated characteristics.</a> The International Journal of Artificial Organs. 35 (2), 93-107 (2012).</li><li>de Soure, A. M., Fernandes-Platzgummer, A., Silva, d. a., L, C., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+microcarrier-based+manufacturing+of+mesenchymal+stem/stromal+cells.">Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.</a> Journal of Biotechnology. 236, 88-109 (2016).</li><li>Rafiq, Q. A., Coopman, K., Nienow, A. W., Hewitt, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+microcarrier+screening+and+agitated+culture+conditions+improves+human+mesenchymal+stem+cell+yield+in+bioreactors.">Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors.</a> Biotechnology Journal. 11 (4), 473-486 (2016).</li><li>Tavassoli, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+production+of+stem+cells+utilizing+microcarriers:+A+biomaterials+engineering+perspective+from+academic+research+to+commercialized+products.">Large-scale production of stem cells utilizing microcarriers: A biomaterials engineering perspective from academic research to commercialized products.</a> Biomaterials. 181, 333-346 (2018).</li><li>Mizukami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+for+large-scale+umbilical+cord-derived+MSC+expansion:+Experimental+performance+and+cost+of+goods+analysis.">Technologies for large-scale umbilical cord-derived MSC expansion: Experimental performance and cost of goods analysis.</a> Biochemical Engineering Journal. 135, 36-48 (2018).</li><li>Yan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersible+and+dissolvable+porous+microcarrier+tablets+enable+efficient+large-scale+human+mesenchymal+stem+cell+expansion.">Dispersible and dissolvable porous microcarrier tablets enable efficient large-scale human mesenchymal stem cell expansion.</a> Tissue Engineering. Part C, Methods. 26 (5), 263-275 (2020).</li><li>Carletti, E., Motta, A., Migliaresi, C., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+for+tissue+engineering+and+3D+cell+culture.">Scaffolds for tissue engineering and 3D cell culture.</a> 3D Cell Culture: Methods and Protocols. , 17-39 (2011).</li><li>Loh, Q. L., Choong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+scaffolds+for+tissue+engineering+applications:+Role+of+porosity+and+pore+size.">Three-dimensional scaffolds for tissue engineering applications: Role of porosity and pore size.</a> Tissue Engineering. Part B Reviews. 19 (6), 485-502 (2013).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMP-grade+microcarrier+and+automated+closed+industrial+scale+cell+production+platform+for+culture+of+MSCs.">GMP-grade microcarrier and automated closed industrial scale cell production platform for culture of MSCs.</a> Journal of Tissue Engineering and Regenerative Medicine. 16 (10), 934-944 (2022).</li><li>NMPA. Pharmaceutical Excipient Registration Database: Microcarrier tablets for cells. Center for Drug Evaluation Available from: <a target="_blank" href="https://www.cde.org.cn/main/xxgk/listpage/ba7aed094c29ae314670a3563a716e">https://www.cde.org.cn/main/xxgk/listpage/ba7aed094c29ae314670a3563a716e</a> (2023)</li><li>List of Drug Master Files (DMFs). US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/drug-mater-files-dmfs/list-drug-master-files-dmfs">https://www.fda.gov/drug-mater-files-dmfs/list-drug-master-files-dmfs</a> (2023)</li><li>Beeravolu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mesenchymal+stromal+cells+from+human+umbilical+cord+and+fetal+placenta.">Isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta.</a> Journal of Visualized Experiments. (122), e55224 (2017).</li><li>Xie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quality+evaluation+system+establishment+of+mesenchymal+stromal+cells+for+cell-based+therapy+products.">The quality evaluation system establishment of mesenchymal stromal cells for cell-based therapy products.</a> Stem Cell Research &amp; Therapy. 11 (1), 176 (2020).</li><li>Maillot, C., Sion, C., De Isla, N., Toye, D., Olmos, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+by+design+to+define+critical+process+parameters+for+mesenchymal+stem+cell+expansion.">Quality by design to define critical process parameters for mesenchymal stem cell expansion.</a> Biotechnology Advances. 50, 107765 (2021).</li><li>Silva Couto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+human+mesenchymal+stem/stromal+cells+(hMSCs)+in+bioreactors+using+microcarriers:+Lessons+learnt+and+what+the+future+holds.">Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: Lessons learnt and what the future holds.</a> Biotechnology Advances. 45, 107636 (2020).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+bead-to-bead+cell-transfer+method+for+serial+subculture+and+large-scale+expansion+of+human+mesenchymal+stem+cells+in+bioreactors.">Facile bead-to-bead cell-transfer method for serial subculture and large-scale expansion of human mesenchymal stem cells in bioreactors.</a> Stem Cells Translational Medicine. 10 (9), 1329-1342 (2021).</li><li>Tsai, A. C., Pacak, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprocessing+of+human+mesenchymal+stem+cells:+From+planar+culture+to+microcarrier-based+bioreactors.">Bioprocessing of human mesenchymal stem cells: From planar culture to microcarrier-based bioreactors.</a> Bioengineering. 8 (7), 96 (2021).</li><li>Hewitt, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+human+mesenchymal+stem+cells+on+microcarriers.">Expansion of human mesenchymal stem cells on microcarriers.</a> Biotechnology Letters. 33 (11), 2325-2335 (2011).</li><li>Mawji, I., Roberts, E. L., Dang, T., Abraham, B., Kallos, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+opportunities+in+downstream+separation+processes+for+mesenchymal+stromal+cells+cultured+in+microcarrier-based+stirred+suspension+bioreactors.">Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors.</a> Biotechnology and Bioengineering. 119 (11), 3062-3078 (2022).</li><li>Schirmaier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-up+of+adipose+tissue-derived+mesenchymal+stem+cell+production+in+stirred+single-use+bioreactors+under+low-serum+conditions.">Scale-up of adipose tissue-derived mesenchymal stem cell production in stirred single-use bioreactors under low-serum conditions.</a> Engineering in Life Sciences. 14 (3), 292-303 (2014).</li><li>Lawson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+development+for+expansion+of+human+mesenchymal+stromal+cells+in+a+50L+single-use+stirred+tank+bioreactor.">Process development for expansion of human mesenchymal stromal cells in a 50L single-use stirred tank bioreactor.</a> Biochemical Engineering Journal. 120, 49-62 (2017).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+microcarrier+culture+of+HEK293T+cells+and+Vero+cells+in+single-use+bioreactors.">Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors.</a> AMB Express. 9 (1), 70 (2019).</li><li>Fang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+scalable+vaccinia+virus-based+vector+production+process+using+dissolvable+porous+microcarriers.">Development of scalable vaccinia virus-based vector production process using dissolvable porous microcarriers.</a> 25th Annual Meeting of the American Society of Gene &amp; Cell Therapy. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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&#38;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&#160; 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&#160; 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); 
			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&#160;</td>
			<td >200-074-400</td>
			<td >Used for expanding the 3D FloTrix vivaPACK Disposable Fill&#38;Finish Consumable Kit.</td>
		</tr>
		<tr >
			<td >Dimethyl Sulfate (DMSO)&#160;</td>
			<td >Sigma</td>
			<td >D2650-100mL</td>
			<td >Used for preparation of cryopreservation solution.</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;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&#160;</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&#39;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&#39;-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

2.9K Views.  Tsinghua University. 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.

Video: Author Spotlight: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

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 ...

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 ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

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 ...