* Corresponding Author: Bruce A. Bunnell, Ph.D., Center for Gene Therapy, Department of Pharmacology, Division of Gene Therapy, Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA 70433, Phone: 985-871-6594, Fax: 985-871-6564, ude.enalut@llennubb
The publisher's final edited version of this article is available at MethodsThe emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in Adipose-derived Stem Cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.
Keywords: Adipose-derived stem cells (ASCs), Biopsy, Differentiation, Expansion, Isolation, Lipoaspirate, Mesenchymal Stem Cells (MSCs)
By definition, a stem cell is characterized by its ability to undergo self-renewal and its ability to undergo multilineage differentiation and form terminally differentiated cells. Ideally, a stem cell for regenerative medicinal applications should meet the following set of criteria: (i) should be found in abundant quantities (millions to billions of cells); (ii) can be collected and harvested by a minimally invasive procedure; (iii) can be differentiated along multiple cell lineage pathways in a reproducible manner; (iv) can be safely and effectively transplanted to either an autologous or allogeneic host [1].
Tissue specific stem cells comprise the second group and are derived from specific organs, such as brain, gut, lung, liver, adipose tissue and bone marrow [2]. It has become evident that these stem cells persist in adult tissues, although they represent a rare population localized in small niches [3]. Postnatal (adult) stem cells are not totipotent; however, they are pluripotent. These cells retain a broad differentiation potential but their developmental potential is more restricted than embryonic stem cells. Adult stem cells were initially thought to have the differentiation capacity limited to their tissue of origin, however recent studies have demonstrated that stem cells have the capacity to differentiate into cells of mesodermal, endodermal and ectodermal origins [4–10]. The plasticity of MSCs most often refers to the inherent ability retained within stem cells to cross lineage barriers and to adopt the phenotypic, biochemical and functional properties of cells unique to other tissues. Adult mesenchymal stem cells can be isolated from bone marrow and adipose tissue. With the increased incidence of obesity in the U.S. and abroad, subcutaneous adipose tissue is abundant and readily accessible [11]. Approximately 400,000 liposuction surgeries are performed in the U.S. each year and these procedures yield anywhere from 100 ml to >3 liters of lipoaspirate tissue [12]. This material is routinely discarded. As discussed below, adipose-derived stem cells are multipotent and hold promise for a range of therapeutic applications.
Adipocytes develop from mesenchymal cells via a complex cascade of transcriptional and non-transcriptional events that occurs throughout human life. Stromal cells that have preadipocyte characteristics can be isolated from adipose tissue of adult subjects, propagated in vitro and induced to differentiate into adipocytes [13–16]. Adipocyte differentiation is a complex process accompanied by coordinated changes in cell morphology, hormone sensitivity and gene expression that have been studied primarily in murine preadipocyte cell lines rather than in human preadipocytes. This protocol describes primary in vitro culture of stromal cell isolated from either large or small quantities of human adipose tissue. While historically in the literature adipose-derived stromal cells have been termed “pre-adipocytes” [14, 15], there is a growing appreciation that they are multipotent, with chondrogenic, neurogenic and osteogenic capability [14, 15, 17–19].
A variety of names have been used to describe the plastic adherent cell population isolated from collagenase digests of adipose tissue. The following terms have been used to identify the same adipose tissue cell population: Adipose-derived Stem/Stromal Cells (ASCs); Adipose Derived Adult Stem (ADAS) Cells, Adipose Derived Adult Stromal Cells, Adipose Derived Stromal Cells (ADSC), Adipose Stromal Cells (ASC), Adipose Mesenchymal Stem Cells (AdMSC), Lipoblast, Pericyte, Pre-Adipocyte, Processed Lipoaspirate (PLA) Cells. The use of this diverse nomenclature has lead to significant confusion in the literature. To address this issue, the International Fat Applied Technology Society reached a consensus to adopt the term “Adipose-derived Stem Cells” (ASCs) to identify the isolated, plastic-adherent, multipotent cell population. The ASCs have been distinguished from the plastic adherent adult stem/progenitor cells from bone marrow originally referred to as fibroblastoid colony forming units, then in the hematological literature as marrow stromal, subsequently as mesenchymal stem cells, and most recently as multipotent mesenchymal stromal cells (MSCs).
The initial methods to isolate cells from adipose tissue were pioneered by Rodbell and colleagues in the 1960s [20–22]. They minced rat fat pads, washed extensively to remove contaminating hematopoietic cells, incubated the tissue fragments with collagenase and centrifuged the digest, thereby separating the floating population of mature adipocytes from the pelleted stromal vascular fraction (SVF) ( Figure 1 ). The SVF consisted of a heterogeneous cell population, including circulating blood cells, fibroblasts, pericytes and endothelial cells as well as “pre-adipocytes” or adipocyte progenitors [20–22]. The final isolation step selected for the plastic adherent population within the SVF cells, which enriched for the “pre-adipocytes”. Subsequently, this procedure has been modified for the isolation of cells from human adipose tissue specimens [23–27]. Initially, fragments of human tissue were minced by hand; however, with the development of liposuction surgery, this procedure has been simplified. During tumescent liposuction, plastic surgeons infuse the subcutaneous tissues with a saline solution containing anesthetic and/or epinephrine via a cannula and then remove both the liquid and tissue under suction [28]. The procedure generates finely minced tissue fragments whose size depends on the cannula’s dimensions. Independent studies have determined that liposuction aspiration alone does not significantly alter the viability of isolated SVF cells [29–31]. Indeed, adherent stromal cells with characteristics of adipocyte progenitors can be found directly within the liposuction aspiration fluid, as well as in SVF derived from the tissue fragment digests [32]. However, when ultrasound-assisted liposuction is performed, the number of cells recovered from tissue digests is reduced, as is their proliferative capacity [31]. The recovery of ASCs can be improved further by manipulating the centrifugation speed [33]. Investigators have achieved optimal cell recovery using a centrifugation speed of 1200 X g based on the subsequent formation of a human-derived adipose tissue depot following implantation in an immunodeficient murine model [33].
Scheme for processing of adipose tissue and isolation of adipose-derived stem cells.
Adipose tissue is collected by needle biopsy or liposuction aspiration. The adipose sample can be kept at room temperature for no more than 24 hours prior to use ( Figure 1 ). ASCs can be isolated from adipose tissue by first washing the tissue sample extensively with phosphate-buffered saline (PBS) containing 5% Penicillin/Streptomycin (P/S). Upon removal of debris, the sample is placed in a sterile tissue culture plate with 0.075% Collagenase Type I prepared in PBS containing 2% P/S for tissue digestion. Mince the adipose tissue sample using two scalpels and pipette the sample up and down with a 25 or 50 ml pipette several times to further facilitate the digestion. Incubate the sample for 30 min at 37 °C, 5% CO2, and then neutralize the Collagenase Type I activity by adding 5 ml of α-MEM containing 20% heat inactivated fetal bovine serum (FBS, Atlanta Biological, Atlanta, GA),) to the tissue sample. Pipette the sample up and down several times to further disintegrate aggregates of the adipose tissue. Upon disintegration, transfer the sample to a 50 ml tube, avoiding the solid aggregates. The stromal vascular fraction (SVF), containing the ASCs, is obtained by centrifuging the sample at 2000 rpm for 5 min. Take the samples out of the centrifuge and shake them vigorously to thoroughly disrupt the pellet and to mix the cells. This step completes the separation of the stromal cells from the primary adipocytes. Repeat the centrifugation step. After spinning, aspirate all the collagenase solution above the pellet without disturbing the cells. The pellet is then resuspended in 1 ml lysis buffer, incubated for 10 min on ice, washed with 20 ml of PBS/2% P/S and centrifuged at 2000 rpm for 5 min. The supernatant is aspirated, the cell pellet is resuspended in a maximum of 3 ml of stromal medium (alpha-MEM, Mediatech, Herndon, VA) supplemented with 20% FBS, 1% L-glutamine (Mediatech), and 1% penicillin/streptomycin (Mediatech) and the cell suspension is filtered through 70 µm cell strainer. It is important to note that all fetal bovine serum should be pre-screened prior to purchase for its ability to support both cell proliferation and adipocyte differentiation. Wash the cell strainer with an additional 2 ml of stromal medium to obtain any additional cells. Plate the sample containing the cells in a lysine coated culture plate and incubate at 37 °C, 5% CO2. Inoculate the cells in a single well of a 12 well plate for an amount of about 500 mg of adipose tissue or in a single well of a 24 well plate for an amount of 250 to 150 mg of adipose tissue.
Seventy-two hours after plating, aspirate the entire medium from the wells. Again it is essential to note that the percent of preadipocytes obtained from the stromal vascular fraction after digestion is patient-dependent. If the SVF does not expand well, increase the FBS contained in the stromal medium to 25%; however this may promote premature adipogenesis. Signs of deterioration such as granularity around the nucleus, cytoplasmic vacuolations and/or detachment of the cells from the plastic surface may indicate inadequate or toxic medium, microbial contamination or senescence of the primary cells.
Wash the cells with prewarmed PBS (1% antibiotic can be added to the solution). Pipette the solution over the cell layer several times in order to clean the cells thoroughly from any tissue fragments and/or blood cells. Add a volume of fresh stromal medium according to the well capacity of the culture plate. The cells are maintained in a humidified tissue culture incubator at 37 °C with 5% CO2. Medium is changed every second day until the cells reach 80–90% confluence, and then there are two options: either harvest the cells or directly induce the adipocyte differentiation. For harvesting viable ASCs, add a small volume (250–500 µl) of sterile warm PBS to the wells and allow PBS to remain on cells for 2 min. Replace the PBS with 500 µl of Trypsin/EDTA solution (0.5%). Incubate in incubator for 5–10 min. Verify under microscope that more than 90% of the cells have detached and then add 500 µl of stromal medium to allow the serum contained in the solution to neutralize the trypsin reaction. Transfer the medium containing the suspended cells from the well to a sterile 2 ml tube. Centrifuge at 1200 rpm for 5 min. Aspirate the supernatant and suspend the cells in a small volume of stromal medium (~250 µl). Proceed to cell counting by taking an aliquot of cells diluted in trypan blue (for a 1:8 dilution: add 12.5 µl of suspended cells to 87.5 µl of trypan blue). Count cells using the hemocytometer. After counting, cells can then be replated according to the well capacity in adequate cell culture plates.
When expanded from a frozen vial, the cells are rapidly thawed at 37 °C and immediately seeded in a 28 cm 2 or a 175 cm 2 plate with complete stromal medium. Medium is changed the day after seeding and then every second day.
ASCs should be harvested at 80–90% confluence for freezing. To collect cells, remove the culture medium and replace with a small volume of sterile, warm PBS. After two minutes, remove the PBS and replace with trypsin-EDTA solution. Incubate the culture dish at 37 °C for at least 5 minutes, or until approximately 90% of the cells have detached from the bottom of the dish. Progress can be monitored under a microscope; the treated cells will be rounded and floating. Add an equal volume of stromal medium to inactivate the trypsin, and transfer the suspension to a conical centrifuge tube. Centrifuge the tube at 1200 rpm for 5 minutes to pellet the cells. Carefully remove the supernatant, and resuspend the cell pellet in 1–2 ml of room temperature cryopreservation medium. Cryopreservation medium consists of 80% fetal bovine serum, 10% dimethylsulfoxide (DMSO) and 10% DMEM/Ham’s F-12, and should be used within two weeks of its preparation. Determine the cell concentration, and then dilute to a final concentration of 2 million viable cells per milliliter of cryopreservation medium. Aliquot the suspension into labeled cryovials, 1 ml/vial. Place the vials in an alcohol freezing container, and store at −80 °C overnight. The freezing container will cool the vials slowly, at approximately 1°C every minute, until they reach −80 °C. The next day, the frozen vials can be transferred to a liquid nitrogen container for long-term storage. Vials of cells should always be stored in the vapor phase of the LN2 tank.
ASCs display multipotency, meaning they retain the ability to differentiate into cell types of multiple different lineages ( Figures 2 and and3). 3 ). The multipotency of these cells has generated interest in their potential therapeutic value for regenerative medicine. Differentiation of these cells can be directed by the addition of specific cocktails of chemical inducers or cytokines. The differentiation process is patient dependent. The age of the donor can be a factor, since some studies suggest that the differentiation capacity is higher in culture from younger subjects compared to older people.
