The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering

Pytlík, Robert; Stehlík, D; Soukup, Tomáš; Kalbáčová, Marie Hubálek; Rypáček, František; Trč, Tomáš; Mulinková, Katarína; Michnová, Petra; Kideryová, L; Živný, J; Klener, Pavel; Veselá, Romana; Trněný, Marek

doi:10.1016/j.biomaterials.2009.03.001

Cited by 35 publications

(27 citation statements)

References 54 publications

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“…conducted in order to make use of pluripotent stem cells Xenotransplantation Using Bone for bone regeneration and, with regard to bone regeneraDifferentiation-Induced Mouse iPS Cells tion, studies have been conducted using ES cells (16) and bone marrow MSCs (29). In particular, differentiaBone differentiation-induced mouse iPS cells were transplanted into 12-week-old Wistar rats (n = 3), at 1 × tion of MSCs is comparatively easy to regulate and these cells are thus being applied clinically.…”

Section: Ips Cells Facilitates Regeneration Radiological and Histologmentioning

confidence: 99%

Transplantation of Osteogenically Differentiated Mouse iPS Cells for Bone Repair

et al. 2012

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Induced pluripotent stem (iPS) cells are a type of undifferentiated cell that can be obtained from differentiated cells and have the pluripotent potential to differentiate into the musculoskeletal system, the myocardium, vascular endothelial cells, neurons, and hepatocytes. We therefore cultured mouse iPS cells in a DMEM containing 15% FBS, 10 −7 M dexamethasone, 10 mM β-glycerophosphate, and 50 µg/ml ascorbic acid for 3 weeks, in order to induce bone differentiation, and studied the expression of the bone differentiation markers Runx2 and osteocalcin using RT-PCR in a time-dependent manner. Osteocalcin, a bone differentiation marker in bone formation, exhibited the highest expression in the third week. In addition, the deposition of calcium nodules was observed using Alizarin red S staining. iPS cells cultured for bone differentiation were transplanted into severe combined immunodeficiency (SCID) mice, and the osteogenic potential exhibited after 4 weeks was studied. When bone differentiation-induced iPS cells were transplanted into SCID mice, bone formation was confirmed in soft X-ray images and tissue specimens. However, teratoma formation was confirmed in 20% of the transplanted models. When mouse iPS cells were treated with irradiation of 2 Gray (Gy) prior to transplantation, teratoma formation was inhibited. When mouse iPS cells treated in a likewise manner were xenotransplanted into rats, bone formation was confirmed but teratoma formation was not observed. It is believed that irradiation before transplantation is an effective way to inhibit teratoma formation.

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Section: Ips Cells Facilitates Regeneration Radiological and Histologmentioning

confidence: 99%

Transplantation of Osteogenically Differentiated Mouse iPS Cells for Bone Repair

et al. 2012

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“…Human mesenchymal stromal cells are progenitor cells that can differentiate into different cell types such as osteoblasts, adipocytes, or chondrocytes. 1,2 Historically, hMSCs were primarily isolated from bone marrow. 3 Currently, other sources of hMSCs are also available, including adipose tissue, 4 dental pulp, 5 and fetal sources such as umbilical cord blood, placenta, and Wharton jelly.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

Verdanova

Pytlik

Kalbáčová

2014

Biopreservation and Biobanking

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A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBSfree medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

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“…Superiority of alpha-MEM over DMEM with respect to MSC expansion was found also in our own unpublished experiments. There is a number of expansion media claimed to be GMP compliant: the LP02 basic medium (Lange et al, 2007), or the CellGro TM medium for hematopoietic stem cells (Pytlík et al, 2009). However, both of these need to be supplemented with fetal calf serum or some of its human alternatives (see below).…”

Section: Cultivation Vessels and Systemsmentioning

confidence: 99%

“…The only manipulation was addition of supplements three times during the two week cultivation period. MSCs cultivated in this medium had phenotype comparable with MSCs cultivated in alpha-MEM + fetal calf serum and were able to differentiate to three mesodermal lineages (Pytlík et al, 2009). During futher development, we successfully transferred this technology to RoboFLASKs TM (Corning) with silicon rubber seal, which was only three times perforated by blunted needle.…”

Section: An Example Of Msc Cultivation With Cytokine-suplemented Mediummentioning

confidence: 99%

Production of Clinical Grade Mesenchymal Stromal Cells

Pytlík¹,

Slanař²,

Stehlík³

et al. 2011

Regenerative Medicine and Tissue Engineering - Cells and Biomaterials

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The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering

Cited by 35 publications

References 54 publications

Transplantation of Osteogenically Differentiated Mouse iPS Cells for Bone Repair

Transplantation of Osteogenically Differentiated Mouse iPS Cells for Bone Repair

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

Production of Clinical Grade Mesenchymal Stromal Cells

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