Rapid Large-Scale Expansion of Functional Mesenchymal Stem Cells from Unmanipulated Bone Marrow Without Animal Serum

Abstract

Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of > 2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.

Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells

“…Thus, an important challenge in immunotherapy is to improve the replicative capacity of MSCs. In the past 10 years, several methods have been developed for MSC isolation/expansion in vitro, such as using flow cytometry or mononuclear cell gravity sedimentation for isolation (32,33), and using magnetic nanoparticles or basement membrane extracellular matrix, together with culture medium supplementation with different growth factors for expansion (34,35). Although …”

Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions

“…RNA from at least 3¥10 6 MSC was extracted using TRIZOL (Invitrogen, Carlsbad, CA, USA) followed by RNeasy clean-up (QIAGEN), as previously described. 22 RNA quality was controlled using the RNA 6000 Pico LabChip kit (Agilent, Waldbronn, Germany) and quantified with a NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, USA).…”

“…5 The first steps of MSC preparation can be performed without the need for density gradient centrifugation either by red blood cell lysis or by seeding untreated bone marrow aspirate. 6,7 Usually, MSC are separated by exploiting their property of adhering to plastic, while non-adherent cells are removed by washing steps. A more standardized method for the isolation of MSC can be facilitated by a two-step protocol in which cells are seeded at a low cell density of only 10 cells per cm 2 .…”

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

“…The dosage of hMSC in therapeutic applications depends on the type of indication and is generally not well defined. In adults the minimal therapeutic hMSC dose is assessed with 1 -2 · 10 6 hMSC per kg [10]. Furthermore, these cells have to be highly viable.…”