Elsevier

Cytotherapy

Volume 9, Issue 4, 2007, Pages 328-337
Cytotherapy

The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use

https://doi.org/10.1080/14653240701322235Get rights and content

Background

Studies have shown that stem cell therapy could be a novel option for improving neovascularization and cardiac function in patients with ischemic heart disease. Human mesenchymal stromal cells (MSC) have generated wide interest in the clinical setting because of their ability to regenerate tissue. The aim of the study was to test whether freezing and storage of human BM mononuclear cells (BM-MNC) and ex vivo-expanded MSC influenced their phenotypic and functional characteristics as well as proliferation capacity.

Methods

MNC were isolated from BM and divided into two portions: one part was immediately cultured (MSC P0) whereas the second part was frozen for a week before cultivation and analysis (F-MSC P1). Confluent MSC (P0) were harvested and divided: one was analyzed as MSC P1 and the other was frozen for a week before further cultivation and analysis as F-MSC P2.

Results

MSC P1, F-MSC P1 and F-MSC P2 had similar proliferation capacities and demonstrated almost identical expression levels of markers characteristic for MSC. The capacity to form endothelial vascular structures was independent of freezing.

Discussion

The proliferation and differentiation capacity as well as the cellular characteristics were identical in cultivated MSC derived from freshly isolated BM-MNC and MSC derived after freezing and storage of either freshly isolated BM-MNC or ex vivo-cultivated MSC. This highlights the potential clinical use of MSC in patients with cardiac and degenerative diseases, as it would be possible to inject MSC obtained from the same BM aspiration at different time points.

Introduction

Despite improvements in medical and surgical therapy, ischemic heart disease remains the leading cause of heart failure in the western world [1]. Recently, there has been an increasing interest in understanding the biology of adult stem cells. Their use as a source for clinical stem cell therapy is promising, as they have been demonstrated to increase neovascularization of ischemic tissue [2].

The relative easy accessibility of BM and the possibility of autologous transplantation make cells from the BM potential candidates for cell therapy [3]. The BM serves as a reservoir for different classes of stem cells, including mesenchymal stromal cells (MSC) [4]. MSC can be easily isolated from BM, have a high expansion potential, are genetic stable and have reproducible characteristics [5]. Additionally, they are able to participate in the renewal of many different tissues, as they can be induced to differentiate into endothelial cells and cardiomyocytes 6., 7., 8.. Therefore these cells have the potential to induce both myogenesis and angiogenesis when transplanted after a myocardial infarction, chronic myocardial ischemia or heart failure [9].

Recently, it has been shown that injection of culture-expanded MSC from the BM into the ischemic model improved blood flow and cardiac function in the pig [9, 10], rat [11] and mouse heart [12, 13]. In addition, MSC implantation induced therapeutic angiogenesis in a rat model of hind limb ischemia through vascular endothelial growth factor (VEGF) production [14]. However, little information is available regarding the therapeutic potency of MSC transplantation in clinic trials in patients with heart diseases.

Standardization of the methods applied to MSC culturing by good manufacturing practice (GMP) is essential for analysis reproducibility of the outcome. Recently, we have established GMP culture conditions for the isolation, expansion and differentiation of MSC for use in autologous MSC therapy in patients with ischemic heart disease [15]. The aim of the current study was to test whether freezing and storage changed the phenotypic and functional characteristics as well as the proliferation kinetics of MSC derived from freshly isolated BM-MNC or ex vivo-expanded MSC. This question is of great clinical relevance when launching the use of MSC therapy in clinical trials.

Section snippets

BM cell preparation and culture

BM cells were obtained from the iliac crest of 10 healthy volunteers by needle aspiration under local anesthesia. A total of 30 mL BM aspirate was immediately combined with 25 mL RPMI-1640 medium containing GlutaMAX and 25 mm HEPES (GIBCO, Invitrogen GmbH, Lofer, Austria) plus 40 IE heparin/mL (Hospital Pharmacy, Copenhagen, Denmark). The marrow sample was diluted to a total of 1:5 with PBS minus Ca2+ and Mg2+ (137 mm NaCl, 2.7 mm KCl, 1.4 mm NaH2PO4.H2O, 6.5 mm Na2HPO4 2H2O, pH 7.3–7.4) (Hospital

Cell proliferation

When we cultivated freshly isolated BM-MNC or MNC derived from frozen MNC and stored in liquid nitrogen for 1 week, single cells with spindle-shaped morphology appeared 3–4 days after seeding the cells. These MSC rapidly expanded into colonies that became confluent after approximately 3 weeks of ex vivo cultivation. On average, MSC derived from freshly isolated MNC were confluent after 22 days of cultivation and MSC derived from frozen MNC were confluent after 23 days in cultivation (data not

Discussion

Among the many different stem cells, MSC seems to be very promising for clinical therapy because of its ease of accessibility, handling and multilineage potential [3, 17, 18]. The effects seen with clinical stem cell therapy have until now been limited and it has been speculated whether repeated treatments could improve the effects. To carry out sequential treatments, freezing and storage of the cells would be necessary. The present study focused on the effects of freezing and storing freshly

Acknowledgements

The study was supported by the Lundbeck Foundation and the Research Foundation at Rigshospitalet.

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