Chondrogenic potential of hASCs expanded in flask or in a hollow-fiber bioreactor

Pirrone, Cristina; Gobbetti, Alessandra; Caprara, Christian; Bernardini, Giovanni; Gornati, Rosalba; Soldati, Gianni

doi:10.15761/jscrm.1000123

Cited by 7 publications

(5 citation statements)

References 59 publications

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“…Allogeneic cell expansion using the Quantum® bioreactor clearly has the potential to reduce the cost of producing therapeutic cells ex vivo. Many laboratories have demonstrated the utility of the system for the GMP manufacture of large quantities of MSCs derived from the bone marrow, adipose tissues [ 26 – 36 ] and even neural sources [ 48 ]. Quantum® GMP manufacturing protocols have developed from early studies which employed the use of research grade FCS [ 26 , 31 ], to more recent studies that have switched to pooled human platelet lysate for the supplementation of Quantum® cultures [ 28 , 35 , 36 ].…”

Section: Discussionmentioning

confidence: 99%

“…Many laboratories have demonstrated the utility of the system for the GMP manufacture of large quantities of MSCs derived from the bone marrow, adipose tissues [ 26 – 36 ] and even neural sources [ 48 ]. Quantum® GMP manufacturing protocols have developed from early studies which employed the use of research grade FCS [ 26 , 31 ], to more recent studies that have switched to pooled human platelet lysate for the supplementation of Quantum® cultures [ 28 , 35 , 36 ]. Similarly, a shift in the preferred choice of substrate utilised to coat the polysulphone hollow fibres inside the bioreactor has been observed, moving from a recombinant fibronectin source to a cryoprecipitate blood product, as a clinical grade source of fibronectin [ 28 , 32 , 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…There are several publications describing the best practice for the expansion and of the UC-MSC populations using traditional TCP techniques and for the cryogenic banking of UC-MSCs for allogeneic clinical use [ 23 – 25 ]. The good manufacturing practice (GMP) compliant Quantum® bioreactor, manufactured by Terumo BCT, has been used in several pre-clinical and clinical production runs for BM-MSCs and MSCs derived from adipose tissues [ 26 – 36 ], but to our knowledge has not been used to expand UC-MSCs. Sub-populations of cells (distinct in morphology to MSCs) have been described following the initial expansion phase of cells from the bone marrow [ 37 ], but these are poorly characterised.…”

Section: Introductionmentioning

confidence: 99%

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A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells

et al. 2019

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BackgroundThe manufacture of mesenchymal stem/stromal cells (MSCs) for clinical use needs to be cost effective, safe and scaled up. Current methods of expansion on tissue culture plastic are labour-intensive and involve several ‘open’ procedures. We have used the closed Quantum® hollow fibre bioreactor to expand four cultures each of MSCs derived from bone marrow (BM) and, for the first time, umbilical cords (UCs) and assessed extensive characterisation profiles for each, compared to parallel cultures grown on tissue culture plastic.MethodsBone marrow aspirate was directly loaded into the Quantum®, and cells were harvested and characterised at passage (P) 0. Bone marrow cells were re-seeded into the Quantum®, harvested and further characterised at P1. UC-MSCs were isolated enzymatically and cultured once on tissue culture plastic, before loading cells into the Quantum®, harvesting and characterising at P1. Quantum®-derived cultures were phenotyped in terms of immunoprofile, tri-lineage differentiation, response to inflammatory stimulus and telomere length, as were parallel cultures expanded on tissue culture plastic.ResultsBone marrow cell harvests from the Quantum® were 23.1 ± 16.2 × 106 in 14 ± 2 days (P0) and 131 ± 84 × 106 BM-MSCs in 13 ± 1 days (P1), whereas UC-MSC harvests from the Quantum® were 168 ± 52 × 106 UC-MSCs after 7 ± 2 days (P1). Quantum®- and tissue culture plastic-expanded cultures at P1 adhered to criteria for MSCs in terms of cell surface markers, multipotency and plastic adherence, whereas the integrins, CD29, CD49c and CD51/61, were found to be elevated on Quantum®-expanded BM-MSCs. Rapid culture expansion in the Quantum® did not cause shortened telomeres when compared to cultures on tissue culture plastic. Immunomodulatory gene expression was variable between donors but showed that all MSCs upregulated indoleamine 2, 3-dioxygenase (IDO).ConclusionsThe results presented here demonstrate that the Quantum® can be used to expand large numbers of MSCs from bone marrow and umbilical cord tissues for next-generation large-scale manufacturing, without impacting on many of the properties that are characteristic of MSCs or potentially therapeutic. Using the Quantum®, we can obtain multiple MSC doses from a single manufacturing run to treat many patients. Together, our findings support the development of cheaper cell-based treatments.Electronic supplementary materialThe online version of this article (10.1186/s13287-019-1202-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells

et al. 2019

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“…After thawing, SVF cells were culture for cell expansion in flask (Nunc, Thermo Fisher Scientific, USA) at 3x10 3 cells/cm 2 as previously described [27]. The cell expansion medium consisted of 5% pooled human platelet lysate (hPL, Stemulate, Cook Regentec, USA) in α MEM medium without nucleosides with Glutamax (Fisher Scientific, USA) supplemented of 0.1 μg Primocin (InvivoGen, USA).…”

Section: Methodsmentioning

confidence: 99%

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs

et al. 2019

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Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

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“…hASCs are found in the stromal vascular fraction of adipose tissue, which is easily accessible in large quantities by using a minimally invasive harvesting procedure [16]. In addition, hASCs seem to be as effective and, in some cases even more suitable, than bone marrow-derived MSCs in clinical applications [17,18].…”

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confidence: 99%

Recovery Ability of Human Adipose Stem Cells Exposed to Cobalt Nanoparticles: Outcome of Dissolution

Borgese

Rossi

Bonfanti

et al. 2020

Nanomedicine (Lond.)

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Aim:To demonstrate that cobalt nanoparticles doses are safe for use in humans and to understand the consequences of the particulate effects, which may persist inside the cells. Materials & methods: Human adipose stem cells were used. We evaluated cell recovery by viability test, morphology and ultrastructure using electronic and optical microscopy, while gene expression was assessed utilizing real-time PCR. Results: After exposure, most stem cells recovered their normal function. Co 3 O 4 -nanoparticles remained inside the cell for the entirety of the considered time. A slight modification of gene expression was observed in the exposed cells. Conclusion: After exposure to 100 M cobalt nanoparticles, most cells returned to normal function. Nanoparticle toxicity was due to ions released by dissolution as well as from the nanoparticles themselves.Cobalt (Co), along with iron (Fe) and nickel (Ni), is a transition metal belonging to group VIIIb of the periodic table. It is an essential element for cell physiology and animal health; in fact, Co is an important constituent of vitamin B12 and is involved in several cell pathways, such as the metabolism of fats and carbohydrates, the conversion of folate into their active form, protein synthesis and the prevention of demyelination [1]. Co deficiency is quite unusual and potentially lethal; conversely, Co intoxication, caused by excessive levels in the body, occurs more frequently and is dangerous as well [2].Along with Fe and Ni, Co is one of the three naturally occurring magnetic metals; it is able to retain its magnetic properties at high temperatures. Therefore, Co has many applications, such as for energy storage systems and catalytic processes [3]. In particular, Co, in its bulk form, is used for green technologies and is also an integral component in powering electric vehicles, wind and wave generators, solar energy technologies sensors, electrochemistry and magnetic fluids [4,5]. However, at the nanoscale dimension, too, Co is promising for several nanotechnological applications ranging from chemistry to nanomedicine, where it is used as a highly sensitive MRI contrast agent [6]. In this scenario, an issue that should be considered is the exponential growth of production, use and discharge of Co nanoparticles (Co-NPs). Consequently, the human body is very likely to come into contact, intentionally or not, with Co-NPs that may easily enter the body in different ways [7][8][9], encounter cells of different tissues and then act on them by perturbing their physiology [10]. In this perspective, special attention should be paid to the presence of multipotent stem cells, a population residing in almost all human tissues that fulfills important functions, including renewal, repair and remodeling [11]. Among the different stem cell populations, mesenchymal stem cells (MSCs) are very interesting due to their great potential in many medical applications, such as regenerative medicine, bone marrow transplantation and orthopedic injuries. Unfortunately, MSCs are more sensitive ...

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Chondrogenic potential of hASCs expanded in flask or in a hollow-fiber bioreactor

Cited by 7 publications

References 59 publications

A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells

A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs

Recovery Ability of Human Adipose Stem Cells Exposed to Cobalt Nanoparticles: Outcome of Dissolution

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