scholarly journals Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status

2021 ◽  
Vol 12 (2) ◽  
pp. 25
Author(s):  
Francesco Muoio ◽  
Stefano Panella ◽  
Valentin Jossen ◽  
Matias Lindner ◽  
Yves Harder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Stem Cells ◽  
Marker Genes ◽  
Free Medium ◽  
Serum Free ◽  
Cell Densities ◽  
Short Time ◽  
Serum Free Conditions ◽  
Human Adipose Stem Cells

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

scholarly journals An Approach towards a GMP Compliant In-Vitro Expansion of Human Adipose Stem Cells for Autologous Therapies

2020 ◽  
Vol 7 (3) ◽  
pp. 77 ◽  
Author(s):  
Valentin Jossen ◽  
Francesco Muoio ◽  
Stefano Panella ◽  
Yves Harder ◽  
Tiziano Tallone ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Growth ◽  
Serum Free ◽  
Cultivation Systems ◽  
In Vitro Expansion ◽  
Maximum Cell ◽  
Cell Densities ◽  
Serum Free Conditions ◽  
3D Cultivation

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.

scholarly journals Development of a Biodegradable Microcarrier for the Cultivation of Human Adipose Stem Cells (hASCs) with a Defined Xeno- and Serum-Free Medium

2021 ◽  
Vol 11 (3) ◽  
pp. 925
Author(s):  
Francesco Muoio ◽  
Stefano Panella ◽  
Matias Lindner ◽  
Valentin Jossen ◽  
Yves Harder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Culture Medium ◽  
Free Cell ◽  
Adipose Stem Cells ◽  
Cell Culture Medium ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Human Adipose Stem Cells

Stirred single-use bioreactors in combination with microcarriers (MCs) have established themselves as a technology that has the potential to meet the demands of current and future cell therapeutic markets. However, most of the published processes have been performed using fetal bovine serum (FBS) containing cell culture medium and non-biocompatible MCs. This approach has two significant drawbacks: firstly, the inevitable potential risks associated with the use of FBS for clinical applications; secondly, non-biocompatible MCs have to be removed from the cell suspension before implantation, requiring a step that causes loss of viable cells and adds further costs and complications. This study aimed to develop a new platform based on a chemically defined xeno- and serum-free cell culture medium and biodegradable MC that can support the growth of human adipose stem cells (hASCs) while still preserving their undifferentiated status. A specific combination of components and manufacturing parameters resulted in a MC prototype, called “BR44”, which delivered the desired functionality. MC BR44 allows the hASCs to stick to its surface and grow in a chemically defined xeno- and serum-free medium (UrSuppe). Although the cells’ expansion rate was not as high as with a commercial non-biodegradable standard MC, those cultured on BR44 maintained a better undifferentiated status in both static and dynamic conditions than those cultured on traditional 2D surfaces.

scholarly journals Modulation of Human Adipose Stem Cells’ Neurotrophic Capacity Using a Variety of Growth Factors for Neural Tissue Engineering Applications: Axonal Growth, Transcriptional, and Phosphoproteomic Analyses In Vitro

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1939
Author(s):  
Katharina M. Prautsch ◽  
Alexander Schmidt ◽  
Viola Paradiso ◽  
Dirk J. Schaefer ◽  
Raphael Guzman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Neurotrophic Factor ◽  
Axonal Growth ◽  
Cellular Level ◽  
Adipose Stem Cells ◽  
Transcriptional Analysis ◽  
Axonal Outgrowth ◽  
Human Adipose Stem Cells

We report on a potential strategy involving the exogenous neurotrophic factors (NTF) for enhancing the neurotrophic capacity of human adipose stem cells (ASC) in vitro. For this, ASC were stimulated for three days using NTF, i.e., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), NT4, glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF). The resulting conditioned medium (CM) as well as individual NTF exhibited distinct effects on axonal outgrowth from dorsal root ganglion (DRG) explants. In particular, CM derived from NT3-stimulated ASC (CM-NT3-ASC) promoted robust axonal outgrowth. Subsequent transcriptional analysis of DRG cultures in response to CM-NT3-ASC displayed significant upregulation of STAT-3 and GAP-43. In addition, phosphoproteomic analysis of NT3-stimulated ASC revealed significant changes in the phosphorylation state of different proteins that are involved in cytokine release, growth factors signaling, stem cell maintenance, and differentiation. Furthermore, DRG cultures treated with CM-NT3-ASC exhibited significant changes in the phosphorylation levels of proteins involved in tubulin and actin cytoskeletal pathways, which are crucial for axonal growth and elongation. Thus, the results obtained at the transcriptional, proteomic, and cellular level reveal significant changes in the neurotrophic capacity of ASC following NT3 stimulation and provide new options for improving the axonal growth-promoting potential of ASC in vitro.

Low serum and serum-free culture of multipotential human adipose stem cells

Cytotherapy ◽  
2007 ◽  
Vol 9 (7) ◽  
pp. 637-646 ◽  
Author(s):  
A.M. Parker ◽  
H. Shang ◽  
M. Khurgel ◽  
A.J. Katz
Keyword(s):  
Stem Cells ◽  
Adipose Stem Cells ◽  
Serum Free ◽  
Human Adipose Stem Cells ◽  
Low Serum

scholarly journals Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro

2013 ◽  
Vol 4 (1) ◽  
pp. 17 ◽  
Author(s):  
Laura Kyllönen ◽  
Suvi Haimi ◽  
Bettina Mannerström ◽  
Heini Huhtala ◽  
Kristiina M Rajala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Adipose Stem Cells ◽  
Human Adipose Stem Cells

Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cells in vitro

Cytotherapy ◽  
2009 ◽  
Vol 11 (7) ◽  
pp. 958-972 ◽  
Author(s):  
Bettina Lindroos ◽  
Shayne Boucher ◽  
Lucas Chase ◽  
Hannu Kuokkanen ◽  
Heini Huhtala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Media ◽  
Proliferation Rate ◽  
Adipose Stem Cells ◽  
Serum Free

190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM

2014 ◽  
Vol 26 (1) ◽  
pp. 209
Author(s):  
Y. Zhang ◽  
C. Wei ◽  
P.-F. Zhang ◽  
X. Li ◽  
Y.-S. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Adipose Derived Stem Cells ◽  
Free System ◽  
Serum Free ◽  
Induced Pluripotent ◽  
Serum Free Conditions

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.

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