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Bioengineering
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Stem Cell Bioprocessing and Manufacturing
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Stem Cell Bioprocessing and Manufacturing

A special issue of Bioengineering (ISSN 2306-5354). This special issue belongs to the section "Regenerative Engineering".

Deadline for manuscript submissions: closed (31 May 2020) | Viewed by 69062

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Prof. Dr. Joaquim M. S. Cabral

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Guest Editor
Department of Bioengineering and iBB- Institute of Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa , Portugal
Interests: stem cells research for tissue engineering and regenerative medicine; stem cell bioprocessing and manufacturing: development of novel stem cell bioreactors and advanced bioseparation and purification processes
Special Issues, Collections and Topics in MDPI journals
Dr. Cláudia Lobato da Silva

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Guest Editor
Department of Bioengineering and IBB - Institute of Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
Interests: bone marrow niche; expansion of human stem cells; cellular therapies with stem cells; bioreactors for stem cell culture and production of extracellular vesicles
Special Issues, Collections and Topics in MDPI journals
Dr. Maria Margarida Diogo

E-Mail Website
Guest Editor
Department of Bioengineering and iBB - Institute of Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
Interests: the development of scalable platforms for highly controlled expansion and differentiation of human pluripotent stem cells (hPSC), embryonic (hESC), induced pluripotent stem cells (hiPSC); stem cell-based purification strategies; regenerative medicine; drug screening; disease modelling
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The next healthcare revolution will apply regenerative medicines using human cells and tissues. The aim of the regenerative medicine approach is to create biological therapies or substitutes in vitro for the replacement or restoration of tissue function in vivo lost through failure or disease. However, whilst science has revealed the potential, and early products have shown the power of such therapies, there is an immediate and long-term need for expertise with the necessary skills to face the engineering and life science challenges before the predicted benefits in human healthcare can be realized. Specifically, there is a need for the development of bioprocess technology for the successful transfer of laboratory-based practice of stem cell and tissue culture to the clinic as therapeutics through the application of engineering principles and practices. This Special Issue of Bioengineering on Stem Cell Bioprocessing and Manufacturing addresses the central role in defining the engineering sciences of cell-based therapies by bringing together contributions from worldwide experts on stem cell biology and engineering, bioreactor design and bioprocess development, scale-up, and manufacturing of stem cell-based therapies.

Prof. Dr. Joaquim M. S. Cabral
Assoc. Prof. Dr. Cláudia Lobato da Silva
Assist. Prof. Dr. Maria Margarida Diogo
Guest Editors

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Published Papers (11 papers)

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Editorial

Jump to: Research, Review

3 pages, 172 KiB  
Editorial
Stem Cell Bioprocessing and Manufacturing
by Joaquim M.S. Cabral, Cláudia Lobato da Silva and Maria Margarida Diogo
Bioengineering 2020, 7(3), 84; https://doi.org/10.3390/bioengineering7030084 - 31 Jul 2020
Cited by 4 | Viewed by 4011
Abstract
The next healthcare revolution will apply regenerative medicines using human cells and tissues [...] Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)

Research

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18 pages, 1637 KiB  
Article
Lactate-Based Model Predictive Control Strategy of Cell Growth for Cell Therapy Applications
by Kathleen Van Beylen, Ali Youssef, Alberto Peña Fernández, Toon Lambrechts, Ioannis Papantoniou and Jean-Marie Aerts
Bioengineering 2020, 7(3), 78; https://doi.org/10.3390/bioengineering7030078 - 20 Jul 2020
Cited by 11 | Viewed by 5279
Abstract
Implementing a personalised feeding strategy for each individual batch of a bioprocess could significantly reduce the unnecessary costs of overfeeding the cells. This paper uses lactate measurements during the cell culture process as an indication of cell growth to adapt the feeding strategy [...] Read more.
Implementing a personalised feeding strategy for each individual batch of a bioprocess could significantly reduce the unnecessary costs of overfeeding the cells. This paper uses lactate measurements during the cell culture process as an indication of cell growth to adapt the feeding strategy accordingly. For this purpose, a model predictive control is used to follow this a priori determined reference trajectory of cumulative lactate. Human progenitor cells from three different donors, which were cultivated in 12-well plates for five days using six different feeding strategies, are used as references. Each experimental set-up is performed in triplicate and for each run an individualised model-based predictive control (MPC) controller is developed. All process models exhibit an accuracy of 99.80% ± 0.02%, and all simulations to reproduce each experimental run, using the data as a reference trajectory, reached their target with a 98.64% ± 0.10% accuracy on average. This work represents a promising framework to control the cell growth through adapting the feeding strategy based on lactate measurements. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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23 pages, 6956 KiB  
Article
An Approach towards a GMP Compliant In-Vitro Expansion of Human Adipose Stem Cells for Autologous Therapies
by Valentin Jossen, Francesco Muoio, Stefano Panella, Yves Harder, Tiziano Tallone and Regine Eibl
Bioengineering 2020, 7(3), 77; https://doi.org/10.3390/bioengineering7030077 - 20 Jul 2020
Cited by 16 | Viewed by 5291
Abstract
Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-based therapies, in-vitro expansion is [...] Read more.
Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-based therapies, in-vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible and economic in-vitro expansion of hASCs for autologous therapies is more problematic because the cell material changes for each treatment. Moreover, cell material is normally isolated from non-healthy or older patients, which further complicates successful in-vitro expansion. Hence, the goal of this study was to perform cell expansion studies with hASCs isolated from two different patients/donors (i.e., different ages and health statuses) under xeno- and serum-free conditions in static, planar (2D) and dynamically mixed (3D) cultivation systems. Our primary aim was I) to compare donor variability under in-vitro conditions and II) to develop and establish an unstructured, segregated growth model as a proof-of-concept study. Maximum cell densities of between 0.49 and 0.65 × 105 hASCs/cm2 were achieved for both donors in 2D and 3D cultivation systems. Cell growth under static and dynamically mixed conditions was comparable, which demonstrated that hydrodynamic stresses (P/V = 0.63 W/m3, τnt = 4.96 × 10−3 Pa) acting at Ns1u (49 rpm for 10 g/L) did not negatively affect cell growth, even under serum-free conditions. However, donor-dependent differences in the cell size were found, which resulted in significantly different maximum cell densities for each of the two donors. In both cases, stemness was well maintained under static 2D and dynamic 3D conditions, as long as the cells were not hyperconfluent. The optimal point for cell harvesting was identified as between cell densities of 0.41 and 0.56 × 105 hASCs/cm2 (end of exponential growth phase). The growth model delivered reliable predictions for cell growth, substrate consumption and metabolite production in both types of cultivation systems. Therefore, the model can be used as a basis for future investigations in order to develop a robust MC-based hASC production process for autologous therapies. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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14 pages, 1027 KiB  
Article
Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates
by Sandra M. Jonsdottir-Buch, Kristbjorg Gunnarsdottir and Olafur E. Sigurjonsson
Bioengineering 2020, 7(3), 75; https://doi.org/10.3390/bioengineering7030075 - 20 Jul 2020
Cited by 3 | Viewed by 4449
Abstract
Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like [...] Read more.
Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL). Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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16 pages, 2017 KiB  
Article
Bioreactor Parameters for Microcarrier-Based Human MSC Expansion under Xeno-Free Conditions in a Vertical-Wheel System
by Josephine Lembong, Robert Kirian, Joseph D. Takacs, Timothy R. Olsen, Lye Theng Lock, Jon A. Rowley and Tabassum Ahsan
Bioengineering 2020, 7(3), 73; https://doi.org/10.3390/bioengineering7030073 - 8 Jul 2020
Cited by 36 | Viewed by 9942
Abstract
Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a [...] Read more.
Human mesenchymal stem/stromal cells (hMSCs) have been investigated and proven to be a well-tolerated, safe therapy for a variety of indications, as shown by over 900 registered hMSC-based clinical trials. To meet the commercial demand for clinical manufacturing of hMSCs, production requires a scale that can achieve a lot size of ~100B cells, which requires innovative manufacturing technologies such as 3D bioreactors. A robust suspension bioreactor process that can be scaled-up to the relevant scale is therefore crucial. In this study, we developed a fed-batch, microcarrier-based bioreactor process, which enhances media productivity and drives a cost-effective and less labor-intensive hMSC expansion process. We determined parameter settings for various stages of the culture: inoculation, bioreactor culture, and harvest. Addition of a bioreactor feed, using a fed-batch approach, was necessary to replenish the mitogenic factors that were depleted from the media within the first 3 days of culture. Our study resulted in an optimized hMSC culture protocol that consistently achieved hMSC densities between 2 × 105–6 × 105 cells/mL within 5 days with no media exchange, maintaining the final cell population doubling level (PDL) at 16–20. Using multiple hMSC donors, we showed that this process was robust and yielded hMSCs that maintained expansion, phenotypic characteristic, and functional properties. The developed process in a vertical-wheel suspension bioreactor can be scaled to the levels needed to meet commercial demand of hMSCs. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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15 pages, 219027 KiB  
Article
Ex-Vivo Stimulation of Adipose Stem Cells by Growth Factors and Fibrin-Hydrogel Assisted Delivery Strategies for Treating Nerve Gap-Injuries
by Katharina M. Prautsch, Lucas Degrugillier, Dirk J. Schaefer, Raphael Guzman, Daniel F. Kalbermatten and Srinivas Madduri
Bioengineering 2020, 7(2), 42; https://doi.org/10.3390/bioengineering7020042 - 5 May 2020
Cited by 15 | Viewed by 5477
Abstract
Peripheral nerve injuries often result in lifelong disabilities despite advanced surgical interventions, indicating the urgent clinical need for effective therapies. In order to improve the potency of adipose-derived stem cells (ASC) for nerve regeneration, the present study focused primarily on ex-vivo stimulation of [...] Read more.
Peripheral nerve injuries often result in lifelong disabilities despite advanced surgical interventions, indicating the urgent clinical need for effective therapies. In order to improve the potency of adipose-derived stem cells (ASC) for nerve regeneration, the present study focused primarily on ex-vivo stimulation of ASC by using growth factors, i.e., nerve growth factor (NGF) or vascular endothelial growth factor (VEGF) and secondly on fibrin-hydrogel nerve conduits (FNC) assisted ASC delivery strategies, i.e., intramural vs. intraluminal loading. ASC were stimulated by NGF or VEGF for 3 days and the resulting secretome was subsequently evaluated in an in vitro axonal outgrowth assay. For the animal study, a 10 mm sciatic nerve gap-injury was created in rats and reconstructed using FNC loaded with ASC. Secretome derived from NGF-stimulated ASC promoted significant axonal outgrowth from the DRG-explants in comparison to all other conditions. Thus, NGF-stimulated ASC were further investigated in animals and found to enhance early nerve regeneration as evidenced by the increased number of β-Tubulin III+ axons. Notably, FNC assisted intramural delivery enabled the improvement of ASC’s therapeutic efficacy in comparison to the intraluminal delivery system. Thus, ex-vivo stimulation of ASC by NGF and FNC assisted intramural delivery may offer new options for developing effective therapies. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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Graphical abstract

16 pages, 6373 KiB  
Article
Impact of Dual Cell Co-culture and Cell-conditioned Media on Yield and Function of a Human Olfactory Cell Line for Regenerative Medicine
by Rachael Wood, Pelin Durali and Ivan Wall
Bioengineering 2020, 7(2), 37; https://doi.org/10.3390/bioengineering7020037 - 12 Apr 2020
Cited by 3 | Viewed by 5737
Abstract
Olfactory ensheathing cells (OECs) are a promising candidate therapy for neuronal tissue repair. However, appropriate priming conditions to drive a regenerative phenotype are yet to be determined. We first assessed the effect of using a human fibroblast feeder layer and fibroblast conditioned media [...] Read more.
Olfactory ensheathing cells (OECs) are a promising candidate therapy for neuronal tissue repair. However, appropriate priming conditions to drive a regenerative phenotype are yet to be determined. We first assessed the effect of using a human fibroblast feeder layer and fibroblast conditioned media on primary rat olfactory mucosal cells (OMCs). We found that OMCs cultured on fibroblast feeders had greater expression of the key OEC marker p75NTR (25.1 ± 10.7 cells/mm2) compared with OMCs cultured on laminin (4.0 ± 0.8 cells/mm2, p = 0.001). However, the addition of fibroblast-conditioned media (CM) resulted in a significant increase in Thy1.1 (45.9 ± 9.0 cells/mm2 versus 12.5 ± 2.5 cells/mm2 on laminin, p = 0.006), an undesirable cell marker as it is regarded to be a marker of contaminating fibroblasts. A direct comparison between human feeders and GMP cell line Ms3T3 was then undertaken. Ms3T3 cells supported similar p75NTR levels (10.7 ± 5.3 cells/mm2) with significantly reduced Thy1.1 expression (4.8 ± 2.1 cells/mm2). Ms3T3 cells were used as feeder layers for human OECs to determine whether observations made in the rat model were conserved. Examination of the OEC phenotype (S100β expression and neurite outgrowth from NG108-15 cells) revealed that co-culture with fibroblast feeders had a negative effect on human OECs, contrary to observations of rat OECs. CM negatively affected rat and human OECs equally. When the best and worst conditions in terms of supporting S100β expression were used in NG108-15 neuron co-cultures, those with the highest S100β expression resulted in longer and more numerous neurites (22.8 ± 2.4 μm neurite length/neuron for laminin) compared with the lowest S100β expression (17.9 ± 1.1 μm for Ms3T3 feeders with CM). In conclusion, this work revealed that neither dual co-culture nor fibroblast-conditioned media support the regenerative OEC phenotype. In our case, a preliminary rat model was not predictive of human cell responses. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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9 pages, 2937 KiB  
Communication
Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products
by Brian Lee, Breanna S. Borys, Michael S. Kallos, Carlos A. V. Rodrigues, Teresa P. Silva and Joaquim M. S. Cabral
Bioengineering 2020, 7(2), 31; https://doi.org/10.3390/bioengineering7020031 - 28 Mar 2020
Cited by 14 | Viewed by 7563
Abstract
Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large [...] Read more.
Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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Graphical abstract

11 pages, 9514 KiB  
Article
Electrospinning Live Cells Using Gelatin and Pullulan
by Nasim Nosoudi, Anson Jacob Oommen, Savannah Stultz, Micah Jordan, Seba Aldabel, Chandra Hohne, James Mosser, Bailey Archacki, Alliah Turner and Paul Turner
Bioengineering 2020, 7(1), 21; https://doi.org/10.3390/bioengineering7010021 - 22 Feb 2020
Cited by 26 | Viewed by 6568
Abstract
Electrospinning is a scaffold production method that utilizes electric force to draw a polymer solution into nanometer-sized fibers. By optimizing the polymer and electrospinning parameters, a scaffold is created with the desired thickness, alignment, and pore size. Traditionally, cells and biological constitutes are [...] Read more.
Electrospinning is a scaffold production method that utilizes electric force to draw a polymer solution into nanometer-sized fibers. By optimizing the polymer and electrospinning parameters, a scaffold is created with the desired thickness, alignment, and pore size. Traditionally, cells and biological constitutes are implanted into the matrix of the three-dimensional scaffold following electrospinning. Our design simultaneously introduces cells into the scaffold during the electrospinning process at 8 kV. In this study, we achieved 90% viability of adipose tissue-derived stem cells through electrospinning. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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Review

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10 pages, 617 KiB  
Review
Culture Time Needed to Scale up Infrapatellar Fat Pad Derived Stem Cells for Cartilage Regeneration: A Systematic Review
by Sam L. Francis, Angela Yao and Peter F. M. Choong
Bioengineering 2020, 7(3), 69; https://doi.org/10.3390/bioengineering7030069 - 4 Jul 2020
Cited by 6 | Viewed by 3533
Abstract
Adipose tissue is a rich source of stem cells, which are reported to represent 2% of the stromal vascular fraction (SVF). The infrapatellar fat pad (IFP) is a unique source of tissue, from which human adipose-derived stem cells (hADSCs) have been shown to [...] Read more.
Adipose tissue is a rich source of stem cells, which are reported to represent 2% of the stromal vascular fraction (SVF). The infrapatellar fat pad (IFP) is a unique source of tissue, from which human adipose-derived stem cells (hADSCs) have been shown to harbour high chondrogenic potential. This review aims to calculate, based on the literature, the culture time needed before an average knee articular cartilage defect can be treated using stem cells obtained from arthroscopically or openly harvested IFP. Firstly, a systematic literature review was performed to search for studies that included the number of stem cells isolated from the IFP. Subsequent analysis was conducted to identify the amount of IFP tissue harvestable, stem cell count and the overall yield based on the harvesting method. We then determined the minimum time required before treating an average-sized knee articular cartilage defect with IFP-derived hADSCs by using our newly devised equation. The amount of fat tissue, the SVF cell count and the stem cell yield are all lower in arthroscopically harvested IFP tissue compared to that collected using arthrotomy. As an extrapolation, we show that an average knee defect can be treated in 20 or 17 days using arthroscopically or openly harvested IFP-derived hADSCs, respectively. In summary, the systematic review conducted in this study reveals that there is a higher amount of fat tissue, SVF cell count and overall yield (cells/volume or cells/gram) associated with open (arthrotomy) compared to arthroscopic IFP harvest. In addition to these review findings, we demonstrate that our novel framework can give an indication about the culture time needed to scale up IFP-derived stem cells for the treatment of articular cartilage defects based on harvesting method. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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20 pages, 1564 KiB  
Review
Production of Human Pluripotent Stem Cell-Derived Hepatic Cell Lineages and Liver Organoids: Current Status and Potential Applications
by João P. Cotovio and Tiago G. Fernandes
Bioengineering 2020, 7(2), 36; https://doi.org/10.3390/bioengineering7020036 - 9 Apr 2020
Cited by 25 | Viewed by 9264
Abstract
Liver disease is one of the leading causes of death worldwide, leading to the death of approximately 2 million people per year. Current therapies include orthotopic liver transplantation, however, donor organ shortage remains a great challenge. In addition, the development of novel therapeutics [...] Read more.
Liver disease is one of the leading causes of death worldwide, leading to the death of approximately 2 million people per year. Current therapies include orthotopic liver transplantation, however, donor organ shortage remains a great challenge. In addition, the development of novel therapeutics has been limited due to the lack of in vitro models that mimic in vivo liver physiology. Accordingly, hepatic cell lineages derived from human pluripotent stem cells (hPSCs) represent a promising cell source for liver cell therapy, disease modelling, and drug discovery. Moreover, the development of new culture systems bringing together the multiple liver-specific hepatic cell types triggered the development of hPSC-derived liver organoids. Therefore, these human liver-based platforms hold great potential for clinical applications. In this review, the production of the different hepatic cell lineages from hPSCs, including hepatocytes, as well as the emerging strategies to generate hPSC-derived liver organoids will be assessed, while current biomedical applications will be highlighted. Full article
(This article belongs to the Special Issue Stem Cell Bioprocessing and Manufacturing)
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