Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues

Abstract

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits.

Keywords: cytokines; explant culture method; growth factors; human tissue; mesenchymal stem cells; non-enzymatic.

Figure 1

Statistical analysis of mesenchymal stem cell clinical studies published in PubMed until June 2016. A, MSCs have been applied in diverse clinical conditions, mostly including cardiovascular diseases, transplant‐related conditions, neurologic disorders, bone, joint, gingival disorders and dental implant, and liver complications (n=203). B, Application of adult and perinatal tissues other than bone marrow has been increased in clinical studies during recent years (N=196)

Figure 2

Advantages of explant method comparing with enzyme digestion for MSC isolation. A, Enzymatic digestion method for MSC isolation: tissue fragments are treated with proteolytic enzyme(s) to obtain a single cell suspension. After washing steps, single cell suspension is cultured in the culture dish. During this process, cells are completely separated from tissue and endogenous CKs and GFs are also washed away. Therefore, cells are transferred to the culture dish after two types of stress; proteolysis and separation from the tissue (all of its physiological communications are cut suddenly), and without addition of exogenous GFs, risk of failure is too high. B, Explant culture for MSC isolation: tissue piece(s) is placed in the culture dish without any digestion step. Cells are migrating out of the tissue due to filling of wound condition. They are communicating with companion tissue and cells. Continuation of molecular communication between migrating cells and tissue piece (physiological type of signals and interactions) reduces stress of primary culture; released CKs and GFs from ECM reservoir and cells (of the tissue explant) stimulate cell growth and proliferation without requirement for addition of any exogenous growth factor

Figure 3

Growth factor and cytokine content of human tissues from which mesenchymal stem cells are commonly isolated. Tissues which are used as MSC source produce growth factors and cytokines and store them in the extracellular matrix. These GFs and CKs can be released into the culture medium of the explants primary culture and stimulate growth and proliferation of migrated cells

Figure 4

Comparison of main characteristics of explant‐derived MSCs with those obtained after enzymatic digestion of the original tissues. MSCs which were obtained using explant method were successfully differentiated into mesodermal lineages (osteocytes, adipocytes and chondrocytes),13, 91, 96 they also express stromal markers (CD29, CD44, CD73, CD90, CD105) and are negative for hematopoietic markers (CD34, CD45, HLA‐DR) in a comparable manner to enzymatic‐derived MSCs96; in some cases such as CD44, CD73 and CD90 expression, their level were higher in explant‐derived MSCs, while CD34 and CD45 expression level were lower.13, 115 They showed similar morphology, immunogenicity and immunosuppressive properties. Similar cell yield and doubling time and proliferation rate was also reported for explant‐derived MSCs13, 91, 92