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.

Abbreviations

MSC

mesenchymal stem cell

AD

adipose tissue

BM

bone marrow

DP

dental pulp

HF

hair follicle

UC

umbilical cord

WJ

Wharton's jelly

ASC

adipose‐derived stem cells

SVF

stromal vascular fraction

DMEM

Dulbecco's modified Eagle's medium

α‐MEM

minimum essential medium‐alpha

RPMI

Roswell Park Memorial Institute medium

PBS

phosphate‐buffered saline

FBS

foetal bovine serum

FCS

foetal calf serum

EDTA

ethylenediaminetetraacetic acid

ECM

extracellular matrix

PG

proteoglycan

CK

cytokine

GF

growth factor

VEGF

vascular endothelial growth factor

FGF

fibroblast growth factor

FGFR

fibroblast growth factor receptor

IGF

insulin‐like growth factor

EGF

epidermal growth factor

HGF

hepatocyte growth factor

PDGF

platelet‐derived growth factor

KGF

keratinocyte growth factor

TGFβ

transforming growth factor β

IL

interleukin

CCL

chemokine (CC motif) ligand

CXCL

chemokine (C‐X‐C motif) ligand

HIV

human immunodeficiency virus

PCR

polymerase chain reaction

PBMC

peripheral blood mononuclear cell

MLR

mixed lymphocyte reaction

1. Introduction

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases due to their beneficial characteristics such as stemness and plasticity, lack of alloreactivity and lack of acute adverse reaction.1, 2, 3, 4 One of the most important steps in MSC therapy is their preparation prior to their administration and the first step of preparation is isolation from original tissue. Increasing use of human adult and perinatal tissues for MSC isolation for clinical applications necessitates paying more attention to selection of a proper isolation method. A systematic search in PubMed using search key word “mesenchymal stem cell” and activated filter “clinical trial” obtained 286 results from which 203 were human clinical studies including more than three patients. Statistical analysis of these studies showed application of MSCs in diverse clinical conditions (Figure 1A) and also growing interest towards use of non‐bone marrow human tissues as MSC sources (Figure 1B). MSCs have been isolated from various human tissues such as bone marrow (BM),5 adipose tissue (AD),6 dental pulp (DP),7 hair follicle (HF),8 cornea,9 umbilical cord (UC)10 and placenta.11 Isolation methods were categorized into two major techniques; enzymatic methods and explant culture method. In enzymatic methods, one, two or in some procedures three proteolytic enzymes are used for separation of cells from tissue; the single cell suspension is then cultured in appropriate medium for further cell proliferation. On the other hand in explant method, no enzyme is used; original tissue is excised into smaller pieces which are placed in culture dishes or flasks, and cells then start to migrate out of tissue and adhere to the culture surface.12 These two techniques are different by many aspects such as time to reach isolated cells and economy; however in the case of MSC isolation, it seems that both of them provide acceptable cell yield.13, 14 Explant method possesses several advantages for MSC isolation which is not clearly described in the literature till now. These advantages can make this method as the best choice for MSC isolation in many research and clinical projects. In this review, I focus on molecular events and methodological aspects of explant culture technique for MSC isolation from human adult and perinatal tissues to provide a useful reference for researchers who are going to choose a suitable tissue source and a proper method for MSC isolation, and also for researchers who are interested in optimizing MSC isolation methods. To gather current applicable information, a research project was undertaken to build main parts of the targeted plan: First, advantages of the explant method including presence of the companion tissue in first steps of primary culture and absence of proteolytic enzymes were discussed. Second, explant isolation methods from most common adult (AD, bone marrow, DP, HF and cornea) and perinatal (UC and placenta) MSC sources were searched and were presented in their most intact form that was provided by the researches from the original articles.

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)

2. Advantages of presence of explant tissue pieces in primary culture

In explant culture method, the tissue piece is present during primary culture. The companion of the tissue piece provides some advantages for migrating cells. Although attachment of cells to extracellular matrix (ECM) is not dissociated due to absence of proteolytic enzymes and this is the reason for longer isolation time period; its advantages are not noticed usually. The tissue consists of ECM and cellular components and both of them are active during the process of primary culture (Figure 2) and can exert several advantages as will be discussed below; however, presence of other cell types cannot be troublesome for obtaining pure MSC population because it is limited to primary culture phase. Indeed, unattached cells such as hematopoietic stem cells (in bone marrow sample) will be gradually removed after refreshing the culture media. Adherent non‐stem cells will also be omitted during first subcultures due to their limited ability to proliferate and produce same cells.

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

2.1. Extracellular matrix continues to provide some benefit

In addition to physical scaffolding for the cellular constituents, ECM also provides crucial biochemical and biomechanical signals that are essential for tissue morphogenesis, differentiation and homeostasis. ECM receptors, such as integrins, discoidin domain receptors and syndecans,15, 16, 17 mediate cytoskeletal coupling to the ECM and involve in cell migration through the ECM.18 On the other hand, the ECM constantly remodels and acts dynamically by modification of its molecular components post‐translationally.

Many ECM proteins, including laminins, tenascins, thrombospondins and fibrillins, contain embedded EGF‐like motifs. These structures may bind as anchored ligands to EGF receptors on cells and trigger signals in such a way that is induced by soluble ligands.16, 19, 20 ECM is also a localized reservoir of growth factors and cytokines. Collagens, proteoglycans (PGs), fibronectin, and vitronectin, themselves, or in combination with heparin and heparin sulphate, bind many growth factors, such as fibroblast growth factors (FGFs) and hepatocyte growth factor (HGF);21 they regulate growth factor bioavailability by establishing stable gradients.16 Local release of soluble growth factors from their insoluble anchorage can be induced following proteolytic degradation of ECM proteins or PGs by enzymes such as metalloproteinases22 (evidences for growth factor content of tissues will be discussed widely in the following parts).

Interactions between ECM proteins and growth factors with cell surface receptors stimulate cellular responses through signal transduction and gene transcription regulation. These physical and biochemical characteristics represent a functional model in which the ECM mediates protection by a buffering action and maintains extracellular homeostasis and water retention.22, 23

Stem cell niche plays key roles in stem cell fate24 and ECM is an effective stem cell niche component. Soluble and matrix‐bond effector molecules of ECM interact with cells and regulate their functions and homeostasis between self‐renewal and differentiation.25 Actually extrinsic cues which are provided by the niches’ unique microenvironment and supportive ECM, maintain stemness of stem cells, determine their proliferation rate and specify whether to divide asymmetrically or symmetrically.24 Reduction of instructive cues in injured and aged ECM affects stem cell behaviour.26 Slight variations in ECM characteristics such as altered protein biosynthesis, post‐synthetic modifications or imbalanced proteolytic degradation of ECM components influence stem cell self‐renewal potential.27, 28 Following tissue damage and vascular injury, a wound response stimulates monocyte infiltration to the damaged ECM. Degradation products and cytokines bind to ECM, and monocytes rapidly differentiate into macrophages.29 These activated macrophages, then, secrete several growth factors, cytokines and matrix metalloproteinases.30 The remodelled ECM promotes directional migration of cells towards site of injury.31 In explant method, it is expected that wound response proceeds; cytokines and enzymes are released, and stem cells migrate towards injured parts and out of the tissue. It seems that in explant method, cells start to migrate out of tissue in a process like wound healing and they gradually exit the tissue while they keep their interaction with the tissue explant. If cell migration from tissue pieces occurs in a similar way to wound healing process, it is better to make incisions in the explant tissue piece to facilitate the migration process (Figure 2).

2.2. Cytokines and growth factors are released from the tissue pieces

As discussed earlier, the tissue injury triggers production and release of growth factors by cells in the injured tissue. Therefore, explant tissue pieces release growth factors to the medium due to the activation of wound healing processes in the explant tissues. The tissue offers a cocktail of cytokines and growth factors (not only one, two or three) which can be very expensive when they are added exogenously, especially in large scale. On the other hand, migrated cells may continue their communication with original tissue piece and transduce signals to regulate secretory events in favour of their requirements; therefore, these cytokines and growth factors may be released from the tissue pieces in proper concentrations which are required by migrated cells. The mutual paracrine relationship between migratory cells and tissue piece may continue for a limited time period.

On the other hand, after enzymatic isolation of MSCs, most of the researchers add growth factors as supplement to media during all steps of cell growth and proliferation. They mostly use β‐FGF and platelet‐derived growth factor (PDGF).32, 33, 34, 35, 36, 37 However, in explant culture, the tissue piece can play as a growth factor reservoir during the primary culture.

In this part, I focus on evidences for cytokine and growth factor content of the most common MSC tissue sources (Figure 3).

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

2.2.1. Adipose tissue

Adipose tissue is a source of growth factors such as β‐FGF and vascular endothelial growth factor (VEGF).38, 39 Adipocytes and resident macrophages are the major sources of VEGF in AD.40, 41 Gabrielsson et al. investigated the expression of several FGFs and FGF‐receptors (FGFRs) in human AD and adipose tissue cell fractions obtained from both subcutaneous (sc) and omental (om) depots and detected FGF‐1, FGF‐2 (or β‐FGF), FGF‐7, FGF‐9, FGF‐10 and FGF‐18 transcripts. They found similar expression of FGF‐2, FGF‐7 and FGF‐10 in sc and om AD, and higher levels of FGF‐1 and FGF‐9 in the om AD. Adipocyte and stroma‐vascular (SV) fractions were prepared from both depots and analysed separately for growth factor expression. FGF‐2 was only expressed in adipocytes; FGF‐7, FGF‐9 and FGF‐18 were expressed in the SV fraction; and FGF‐1 and FGF‐10 were expressed in both adipocytes and the SV fraction.42 Currently, the most abundant growth factor that is added to MSC culture medium is FGF‐2 or β‐FGF; therefore, adipocytes can support MSCs’ growth at least by releasing β‐FGF in the primary steps of in vitro culture, and their presence seems to be supportive. Indeed, the presence of SV fraction is not enough for secretory support of MSCs growth and presence of adipocytes in addition to SV fraction in the explant culture can improve in vitro media for stem cell growth and proliferation.

2.2.2. Bone marrow

Bone marrow aspirate contains large quantities of growth factors, cytokines, ECM molecules and cell‐adhesion molecules involving in osteogenesis, hematogenesis, neural growth and angiogenesis.43, 44 Bone marrow nucleated cells secrete angiogenin, VEGF, HGF, insulin‐like growth factor 1 (IGF‐1), transforming growth factors (TGFs) interleukin 10, chemokine (C‐C motif) ligand 2 (CCL2), CCL23 and CCL24, chemokine (C‐X‐C motif) ligand 6 (CXCL6), CXCL12 and CXCL13, and FGF9.45, 46 Specifically, BM‐MSCs were found to secrete many growth factors and cytokines and perform their actions through paracrine and autocrine effect, this characteristic is also of importance in cell therapy purposes such as cardiac and neurologic disorders.47 Secretions from MSCs and other BM cells are present in the aspirated sample and can be very effective in first steps of in vitro cell isolation and primary culture and aid easier adaptation of isolated cells with new environment.

2.2.3. Dental pulp

Growth factors such as VEGF, FGF‐2, PDGF, TGF‐β and HGF were isolated from human DP tissue,48, 49 and they were also found in pulp fibroblast supernatant.50, 51 Both the physiological cell populations of the pulp and the inflammatory cells, which locally infiltrate the tissue at sites of injury, express several growth factors.52 Against dentin matrix in which these growth factors become “fossilized”, in the ECM of the pulp soft tissue, they are in greater turnover, and contribute to the more immediate cellular responses.52 Dentin matrix contains a mixture of cytokines and growth factors which are probably produced by odontoblast cells; they may be released into the pulp environment during tissue injury53 and to the media during primary explant culture of pulp stem cells.

2.2.4. Hair follicle

Production of new hair is a cyclic process in which follicles produce an entire hair shaft from tip to root. When follicle stem cells receive signals through growth factors and cytokines, the next growth phase starts.54 Many growth factors, such as keratinocyte growth factor (KGF) and various FGFs, are released by mesenchymal cells and act as strong mitogens for other follicular cells.55, 56, 57 Kozlowska et al.58 found that cultured follicular cells strongly expressed mRNA of four VEGF molecular species. Other growth factors, such as HGF,59 PDGF,60 FGFs1, 2, 5, 7, 10, 13, 22 and epidermal growth factor (EGF),61, 62, 63 are shown to be expressed in dermal and hair follicular cells to regulate hair growth and skin regeneration.

2.2.5. Cornea

Maintaining homeostasis and normal corneal function and also wound healing response in human cornea is regulated by a number of growth factors and cytokines which is produced by the corneal epithelium, stroma and endothelium.64, 65 Evidences established production of EGF,66 TGF‐β267 and β‐FGF68 by three corneal cell types (stroma, endothelial and epithelial cells). KGF and HGF are believed to be produced by corneal fibroblasts. PDGF is found mainly in the epithelium, IGF in the corneal epithelium and stroma, and the trabecular, meshwork, high levels of aFGF in Bowman's and Descement's membranes and in the corneal endothelium, and bone morphogenic proteins in all layers of cornea. Nerve growth factor, neurotrophin‐3 and ‐4 (NT‐3 and ‐4), and glial cell‐line‐derived neurotrophic factor (GDNF) and (VEGF) have been detected in some studies in the corneal tissue.64, 69

2.2.6. Umbilical cord

Umbilical cord tissue is a source of cytokines and growth factors such as β‐FGF, PDGF, EGF, TGF‐β and IGF‐1. The most abundant is IGF‐1 (almost 350 ng per 1 g of Wharton's jelly). TGF‐β, α‐FGF and β‐FGF also exist in nanograms; however, their amounts are distinctly lower. PDGF and EGF exist in picogram concentrations. The amount of these cytokines in the Wharton's jelly tissue is comparable with or higher than the amount, which exist in the wall of UC artery. Sobolewski et al.70 founded that β‐FGF was released from tissue pieces during primary phase of explant culture in the medium.

2.2.7. Placenta

Implantation and growth of the placenta requires extensive angiogenesis and vascular transformation to establish the structures involved in exchange. For this purpose, growth factors and cytokines are produced and secreted in placenta tissue, which were detected in several investigations. The presence of four species of mRNA encoding VEGF was demonstrated in both first trimester and term placenta.71, 72 VEGF was found to be produced by cells within the villous mesenchyme, decidual macrophages and decidual glands.73 FGF‐2 is also expressed in first trimester and term placenta, its localization was detected in the syncytiotrophoblast surrounding the placental villi and in/around cytotrophoblast cells in first trimester placenta and in the syncytiotrophoblast and foetal membranes at term. FGF‐2 mRNA is localized strongly in the smooth muscle cells around the mid‐ and large‐sized placental vessels.74, 75 Other growth factors are also produced in placenta tissue76 including PDGF in cotyledons,77, 78 HGF in the villous core compartment,79 EGF in cytotrophoblast and syncytiotrophoblast,80 IGF‐1 in cytotrophoblast and syncytiotrophoblast,81 and IGF‐2 in cytotrophoblast.82

3. Absence of proteolytic enzymes in isolation process

3.1. No enzyme/proteolytic stress on cells

Sutradhar et al. showed that trypsin‐EDTA mixture induced a significant loss of both membrane integrity and cell viability in a time‐dependent manner; they investigated most commonly used concentration of trypsin‐EDTA (0.25% trypsin with 0.02% EDTA) on equine chondrocytes (for 5, 20 and 60 minutes) and considered the 5‐minute time period as control. They found that after 20 and 60 minutes of trypsinization, the cell membranes were strongly affected and the percentages of viable cells reduced to 91% and 85% respectively. They suggested that as minimum trypsin exposure time as possible should be applied in the process of cell isolation from tissue.83 Therefore, it is necessary to carefully set up the process of enzymatic cell isolation which may be time‐consuming at first.

Trypsinization can also affect cell surface antigens,84 cell surface topography, cytoskeletal components and intra‐membranous particle distribution.85 These enzyme's toxic effects may revert during several days of culture86; however, in primary culture, it may enhance the probability of isolation failure.

These effects are variable for different tissue samples in each isolation run due to either variability of lytic activity between batches of enzyme and variable sensitivity of tissues (from different persons) to collagenases; therefore, verification of enzymatic isolation method for clinical use is difficult.87

In addition, in explant technique, cells are transferred from in vivo into in vitro condition in companion with a fragment of related tissue; actually they do not suddenly separate from their previous stable environment. This may help cells tolerate isolation stress better; in other words, in enzymatic isolation technique, cells are exposed to dual stress: separation from body and enzymatic interruption. Otte et al.88 demonstrated that in the presence of the originating tissue pieces, the stem cell characteristics of MSCs maintained during long‐term in vitro culture. They suggested that the related tissue fragments provides a microenvironment in which, MSCs retain in their stem cell‐like state.

3.2. No risk of viral transduction through enzyme product

Porcine trypsin is produced using huge numbers of pancreases; in this process, one infected animal may contaminate an entire batch. Although much of bovine serum and trypsin which are used in production of human biologicals are often subjected to virus removal and inactivation procedures, the sensitivity of current tests may not be sufficient to detect diluted contaminants. When sensitivity of tests is not enough for detection of low‐level initial contamination, it can be amplified during the long course of scale‐up. In addition, some viruses—that may have human host—may not be detected by the standard tests and may be of concern as contaminants.89

Pinheiro de Oliveira et al. performed five PCR protocols to assess the presence of genetic material from mycoplasma, porcine circovirus 1, bovine leukaemia virus or bovine viral diarrhoea virus in cell culture samples and demonstrated that sera and trypsin (which were used by different laboratories) showed high rate of contamination. Their results highlighted the necessity of monitoring and controlling of biological contaminants in laboratories and cell banks which are working with these materials.90

3.3. Reduced price

Purchase of enzyme products raises the price in cell isolation experiment (in enzymatic isolation method). In addition, as it was described earlier, explant tissue pieces release cytokines and growth factors into the medium, which lead to cell growth stimulation; so there is no need to add any growth factor. Consequently, simple medium is again more cost‐effective for MSC expansion in both research and clinical settings.

The process of set‐up and design of explant culture method is also less labour‐intensive and more cost‐effective in comparison with enzymatic digestion, so more suitable for clinical purposes.

These facts are of special importance when large‐scale application in clinical settings is the main objective.

4. Technical highlights of explant isolation method for human MSCs

It is necessary to remind that before any human sampling, ethical aspects of the process must be completely achieved. In this part, pivotal technical points of MSC explant isolation from various sources are discussed and fundamental steps of explant primary culture methods for MSC isolation from human tissues are presented in Table 1.

Table 1.

Technical details for MSC isolation from human adult and perinatal tissues by explant primary culture method

Study	Specimen	Preparation	Plating	First medium	First medium change	Explant removal	First observation of migrated cells	Yield
Adipose tissue
91	10 mL Lipoaspirate 5 g resected adipose tissue	Fragments of about 5 mm3	1 g tissue/100 mm dish	2.5 mL knock‐out DMEM + 10% FBS + 2 mmol/L‐glutamine + antibiotics	Not directly mentioned (it seems to be 5‐7 d after plating)	5‐7 d after plating	3‐4 d after plating	5‐8 × 105 cells/g of initial tissue plated (10‐14 d after plating)
92	Small pieces of rat adipose tissue Human lipoaspirate	Small pieces (≈5 mg)	Explanting tissue pieces into the corner of flask	50 μL pre‐warmed FBS	After 24 h DMEM + antibiotic + 20% FBS	3 d after plating	3‐5 d after seeding	NM
93	Inguinal fat pads from 8‐week‐old BALB/c mice	1‐2 mm3 pieces	Spacing between adjacent tissues = 5 mm 5 min for tissue adherence	α‐MEM + 10% FBS	2 d after plating	NM	2 d after plating	24 × 103 cells/mg adipose tissue after 7 d of culture
94	Lipoaspirate	Saline/blood fraction located below the floating, more buoyant adipose tissue	Resuspending the cell pellet in DMEM with 40‐50% FBS followed by plating the cells	DMEM + 40%‐50% FBS	After an overnight incubation DMEM low glucose + 10% FBS + 1% ABAM+ ± 10 ng/mL EGF	After an overnight incubation	NM	~100 000 cells/100 mL of blood/saline collected
Bone marrow
95	Bone marrow sample	Dilution with three equal volumes of growth medium	10 mL of diluted aspirate in each 75‐80 cm2 flask or 10‐cm dish	DMEM low glucose + 10% FBS + antibiotics	Undisturbed at 37°C for 4‐5 d	After 4‐5 d	Small MSC colonies are visible at 5‐7 d	NM
5	Bone marrow sample	Centrifuging cell suspension (1000 rpm, 5 min, at 18°C), resuspending cell pellet in complete medium	Seeding the cell suspension in 25 cm2 flasks (16 × 106 cells/mL)	RPMI‐1640 with 10% FBS and antibiotics	After 6 and 24 h	After 6 and 24 h	Attached cells after 6 h	NM
Dental pulp
97	Extracted third or premolar teeth	Removing pulp tissue from tooth and cutting into small pieces	Explanting pieces on culture surface and covering them by plastic coverslips	DMEM + 2 mmol/L‐glutamine + antibiotics + 20% FBS	After 5 d	NM	NM	NM
96	Third molar teeth	Gentle removal of pulp and mincing into 1‐2 mm3 fragments	Placing pieces in six well plates	α‐MEM + 2 mmol/L l‐glutamine + antibiotics + 10% FBS	NM	NM	2‐4 d	NM
Hair follicle
104	Plucked hairs with full hair follicle	Cutting the root tissue with sterile ophthalmic scissors	Placing hair follicle root tissues into 96‐well plates	DMEM/Ham's DMEM/F12 + 10% FBS + 2 ng/mL bFGF	NM	NM	NM	NM
Cornea
9	Corneal buttons from cadavers	Scrapping epithelium, the Bowman's membrane, then endothelium and the Descemet's membrane, to obtain just central part of cornea and cutting it into small squares	Plating pieces into 24‐well cell culture plates	1 mL low‐glucose DMEM + 10% FCS + 1% antibiotic‐ antimycotic solution	Every alternate day	NM	After 10‐14 d	NM
106	Limbal tissue	Incision and separation of conjunctiva from the limbal junction. Cutting limbal rim into ~1 × 2 × 0.25 mm pieces	Placing each limbal explant with the epithelium side down into a well of 12 well plates	1:1 mixture of DMEM and Ham's F12 medium + 10%/20% human serum + 50 μg/mL gentamicin and penicillin	Every 2‐3 d	Remained for prolonged time period (60‐61 d)	NM	NM
Umbilical cord/Wharton's jelly
109	Umbilical cord	Cutting UC in to 1.5 cm length pieces	Plating one piece/6 cm dish, 5‐10 min for tissue attachment	5 mL of high‐glucose DMEM + 10% FBS + 2 mmol/L l‐glutamine + antibiotics	4 d post‐explantation	Once floating by itself	24 h after plating	NM
109	Umbilical cord	Excising the gelatinous tissue surrounding the vessels and mincing into very fine pieces of 1‐3 mm2	Plating 10‐14 piece/6 cm plate, 5‐10 min for tissue attachment	5 mL of high‐glucose DMEM supplemented with 10% FBS, 2 mmol/L l‐glutamine, and antibiotics	4 d post‐explantation	Once floating by itself	24 h after plating	NM
110	Umbilical cord	Mincing UCs into 1‐2‐mm3 fragments	Aligning 2 g minced UC at regular intervals in 10‐cm culture dishes and covering them with Cellamigo® mesh	α‐MEM supplemented with 10% FBS, gentamicin, and amphotericin B	5 d after plating	Within 9‐12 d	NM	2.9 ± 1.4 × 106 cells/g
Placenta
113	Placenta	Separating maternal deciduas and cutting chorionic villi from foetal portion into 40 mg pieces (wet weight)	Allowing the tissues to adhere to the plastic surface in six‐well plates for 60 min	DMEM‐F12 + 10% FBS + 100 μg/mL l‐glutamate + antibiotics	2 d after plating	14 d after explantation	NM	11.55 × 103 cells/40 mg tissue
114	Placenta	Removing amnion and chorionic plate from placenta and cutting foetal villi into small pieces	Placing tissue pieces into dishes with no coating to attach to the surface	DMEM (low glucose) + 10% FBS + antibiotics/antimycotics	NM	NM	NM	1 × 104 cells/piece after 20 d

NM, not mentioned.

4.1. Adipose tissue

Lipoaspirate tissue and resected AD are appropriate sources of human MSCs. They can be collected from voluntary liposuction and abdominoplasty procedures. Priya et al. could obtain MSCs from both lipoaspirate and fat tissue using explant primary culture method. Cell populations in P0 were enriched in adipose‐derived stem cells (ASCs), because the migration of plastic adherent cells from the tissue pieces served as the criterion for isolation and led to significantly reduced haematopoietic cells and contaminants.91 Ghorbani et al.92 isolated MSCs from AD slightly different; they washed fat tissue pieces with FBS before plating, and then incubated the explanted tissues with very small amount (about 50 μL) of FBS for 24 hours. It seems that this step was performed for better tissue attachment. Jing et al. also isolated MSCs from inguinal fat pads of BALB/c mice. Their procedure was similar with little differences; they stated that tissue pieces should be placed with 5 mm space from each other and after placing them in the flask a 5‐minute period is necessary for tissue attachment before addition of the medium. This time was much shorter than that for other tissues such as mitral valve and skin. This was probably because the released lipids made the minced AD sticky and easy to attach to the flasks.93

In the case of lipoaspirate specimen, fat fraction can be processed by traditional methods; however, blood/saline fraction can also be used to isolate MSCs using a procedure that was performed by Francis et al. They presented a quick method for MSC isolation and expansion from lipoaspirate in <30 minutes, and used only standard tissue culture materials and equipment without using collagenase digestion and Percoll gradients, or multiple washings. They achieved an abundant population of adipose‐derived MSCs with differentiation potential, characteristic cell surface markers and proliferative lifespan mostly same as MSCs extracted from bone marrow (BMSCs) or enzymatically processed ASCs.94

4.2. Bone marrow

The common method for MSC isolation from bone marrow is density gradient centrifugation which was discussed widely in many published studies and is not targeted to be explained again here; however, another method was also examined by researchers in which, the whole tissue was directly plated for primary culture.95 In this method, it is necessary to prepare a single cell suspension by pipetting before plating of sample. Al‐Qaisy et al. compared three methods for MSC isolation from BM and examined direct plating method. They stated that in direct plating, primary tissue culture plate can be contaminated heavily with hematopoietic progenitors such as macrophages and fibroblasts; however, they tried to solve this problem by first using RPMI‐1640 medium which inhibited the growth of hematopoietic cells in cultures, and second frequent media refreshing in 6 and 24 hours of initial culture.5

4.3. Dental pulp

Once deciding to use teeth for the purpose of MSC isolation,96, 97 possibility of contamination should be strictly considered. Laino et al. stated two valuable points in this relation: (i) checking for systemic and oral diseases and pre‐treating a week before with professional dental hygiene, and (ii) covering the dental crown with a 0.3% chlorhexidin gel for 2 minutes, before extraction.98 Perry et al. also recommended to transport the tooth on ice to the laboratory. They also provided a standard washing procedure before starting cell isolation: several washes of tooth in sterile PBS, followed by immersion in 1% povidoneiodine for 2 minutes, then immersion in 0.1% sodium thiosulfate in PBS for 1 minute, and another wash in sterile PBS.99

To remove pulp tissue, the tooth should be broken carefully to avoid pulp damage. For the best, it is recommended to follow Gronthos procedure.100

4.4. Hair follicle

The most difficult step is separation of the follicle from hair. One way is using skin specimens. Skin biopsies can be obtained from different parts of the body, namely from the face, back, forearm, thigh, calf, chest and pubic region. As Ohyama et al.8 stated, microdissecting approaches have been the standard method for obtaining HF; however, other researchers provided various protocols; Krejci et al.101 excised an area under the sebaceous gland because bulge was not a clearly visible structure during preparation. Clewes et al. separated the bulge region completely from other parts by removing the dermal papilla and matrix. They also stated that, for protection against oxidative stress, low oxygen tension was applied for culture condition. They noted that hypoxia is defined as O₂ pressure below the normoxic value in a given tissue and in HFs; oxygen tension ranges between 2.5% and 0.1% O₂.102 Magerl et al. recommended a simplified protocol for isolation of HF; they transected small skin specimens (approximately 1 cm³) at the dermis‐subcutis interface by scalpel to expose the mid‐to‐lower segments of HF embedded in the AD. The upper portion of the HFs from the subcutis was ejected by pressing the sides of the fat tissue with blunt forceps.103 The simplest method was presented by Wang et al. They plucked hair with a full HF from scalp, and simply cut the root tissue with sterile ophthalmic scissors and used the root for explantation.104

4.5. Cornea

Different populations of stem cells have been detected in human corneal tissue.105 Recently, MSC was also isolated from human cornea and found to play role in injury/wound healing process within the tissue.9, 106 Luznic et al. explanted limbal rim pieces including the epithelium as well as some of the superficial limbal stromal tissue. They detected two distinct population of stem cells after long‐term culture (when explants were cultivated for over 30 days) and found that after a second 30‐day explantation of same pieces into another dish, MSC population became 2‐fold in number.106 In another study, Vereb et al. isolated MSCs from just central part of the cornea; by their method, just MSC population proliferated and detected in the culture with no endothelial cell. Their isolation method and culturing condition led to obtaining cells with CD90‐ and CD105‐positive and CD34‐negative markers over 10 passages.9

4.6. Umbilical cord

Mesenchymal stem cells can be isolated from either whole UC107 or Wharton's jelly matrix.108 Although isolated cells from both showed similar properties, the methods are somehow different, especially in considering large‐scale set‐up. Cell isolation from Wharton's jelly is more labour‐intensive than from entire cord piece, because Wharton's jelly must be separated from other parts—umbilical vein and arteries—which is a time‐consuming and labour‐intensive process. On the other hand, in isolation of cells from entire cord piece, there is no need to separate different parts of the cord, and by simple and rapid incisions for better tissue exposure, the tissue can be explanted for primary culture.109

For isolation of MSCs from human UC, Mori et al. used a stainless steel mesh (Cellamigo^®; Tsubakimoto Chain Co. Saitama, Japan.) to prevent tissue explant pieces from floating; they stated that by this improvement, cells can be obtained sooner and with higher yield compared to conventional explant method.110

4.7. Placenta

Due to high probability of transmission of normal flora after vaginal delivery, it is highly recommended to collect placenta after sterile elective caesarean sections, it is also necessary to confirm donor health with no bloodborne infections especially hepatitis, HIV and syphilis.

It is possible to isolate MSCs from foetal or maternal parts of the placenta.111 Amniotic membrane can be isolated from chorion by blunt dissection.112 To isolate MSCs from chorionic villi, maternal deciduas should be separated and discarded, and then chorionic villi should be cut from the foetal portion.113, 114

5. Comparison of MSCs isolated by enzymatic and explant methods

In a number of studies, MSCs were isolated by explant and enzymatic methods from the same tissue and compared for main characteristics. Priya et al. measured surface expression of CD markers (CD34, CD44, CD73, CD90, CD105, HLA‐DR) on MSCs obtained by explant and enzymatic methods at P0, P1, P4, P10. They showed that there were just little differences between MSCs which were isolated by two methods. They also found no difference between immunogenic and immunosuppressive activities of explant and enzymatic‐derived MSC; MSCs did not stimulate proliferation of peripheral blood mononuclear cells (PBMCs), and also strongly suppressed lymphocyte proliferation after mixed lymphocyte (MLR) reaction.91 Explant method led to harvest pure and less heterogeneous cells with higher proliferation rates when compared with enzymatic method13, 96; enzymatic digestion allowed the isolation of fibroblast‐like (stem) cells and also release of endothelial cells and pericytes.96 Salehinejad et al.13 suggested that more homogenous cell population and less enzymatic damages was the reason for higher proliferation rate of MSCs derived by explant method. Significantly lower percentage of HLA‐DR⁺ hematopoietic cells in passage 0 of cells harvested by explant method was again reported by Priya et al.,91 which indicated more homogenous MSC population. Yoon et al.14 also isolated MSCs from Wharton's jelly by explant and enzymatic methods and stated that explant method yielded in higher cell viability and number (2.8 times greater number of cells than enzymatic digestion‐derived MSCs at P0). Jing et al. obtained few cells in the first 2 days post‐explantation; however, the cell number increased very fast in the next days and at last the primary cultured cells obtained by explant and enzymatic methods grew to 80% confluence approximately at the same time. They calculated the nucleated cell number per tissue weight and showed that the explant culture method gave higher yield of stromal cells than the digestion method; may be due to the decreased adhesion ability in the cells exposed to enzymatic treatment, and the loss of cells during the procedures of filtering and washing in the digestion method.93 Shah et al. isolated MSCs from human lipoaspirates by two methods; in the first, they used collagenase and in the second, applied washing steps without any protease. Tissue processing time in non‐enzymatic method was one‐third of the time it took in enzymatic method, and time to reach confluence in enzyme method was less than half of the time for washing method. While primary cell yield was about 19‐fold greater, there was no significant difference according to adipogenic and osteogenic differentiations, and in passage 0, MSCs from both methods showed comparable immunophenotype.115 Jing et al.93 also agreed in shorter processing time in explant culture compared to digestion method; as it took 1 hour to disaggregate the tissue by collagenase. In this regard, Ghorbani et al. pointed that their explant procedure would be a suitable method for isolation of MSCs from small amounts of fat tissue and applicable for patients suffering from cancer and some chronic diseases which are associated with pronounced fat lost (cachexia) and a limited amount of fat can be obtained92 (Figure 4).

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

Cryopreservation is currently the best way for long‐term cell storage; MSCs, isolated by explant method, can be successfully revitalized after cryopreservation while maintaining their characteristics.109, 116 In addition, after cryopreservation of UC tissue, it is possible to isolate MSCs by explant method from post‐thaw tissue pieces.117, 118

6. Research in progress and future directions

One of the most important challenges in preparation of cell therapy products is the application of xenogenic biologicals such as proteolytic enzymes and serum. In MSC isolation and culture, proteolytic enzymes are used during two steps: isolation and subculturing. In explant method, enzymes are omitted from isolation step; however, they are still necessary for passaging. 3D culture systems can be helpful for MSC propagation without application of proteolytic enzymes; however, these systems are still improving.119

For removing animal serum from MSC culture, it is suggested to use human serum obtained from the donor, volunteers or UC blood.120 Human platelet lysate is also recently recommended to be used instead of animal serum.116, 121

Another issue is insufficient cell numbers which was mostly of concern in the case of BM as the most common MSC source. Going towards use of medical waste tissues such as UC and AD, that are available in large amounts, can resolve this problem completely.

Until now explant culture method was applied for isolation of MSCs from few tissue types (which are presented in the current review) as main MSC sources; however, it is highly suggested to apply it for MSC isolation from any other tissue source.

7. Conclusion

Mesenchymal stem cell isolation from human tissues can be performed efficiently using explant culture method. Obtained MSCs possess similar characteristics with those derived by enzyme digestion. However, explant method includes several advantages comparing with enzymatic isolation. These advantages are of significant importance because in explant isolation, proteolytic stress on cells is omitted and they are also in companion with a piece of their tissue origin in the primary culture which increase probability of successful and high yield isolation. Reduced price and risk of biological contaminations are other influential advantages (regarding economic and labour issue). They are satisfactory reasons for using explant methods for MSC isolation in research and clinical settings, against longer time for obtaining cells in primary culture initial step comparing to enzymatic method. Further investigations are recommended to modify explant methods to make this lag time shorter. More experiments can also help us to even better understand the molecular events in primary culture in order to optimize and design isolation step more precisely.

Conflict of interest

There is no conflict of interest. There is no grant or funding support for this manuscript.

Hendijani F. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues. Cell Prolif. 2017;50:e12334. 10.1111/cpr.12334