This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.
Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs — including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.
Mesenchymal stem cells (MSCs) are a class of multipotent adult stem cells that can be isolated from various adult and fetal tissues, including from adipose tissue. These cells are an attractive cell type for both basic research and clinical applications due to their plasticity to differentiate into cells of all germ layers in vitro, cross allogeneic barriers, home to areas of inflammation, and suppress inflammation (reviewed in Sherman, et al.1). Adipose-derived MSCs (ASCs) are particularly appealing due to their ease of obtainment, as adipose tissue is generally considered discard tissue following routine liposuction and abdominoplasty procedures. However, once obtained, the samples are generally subjected to harsh conditions – either enzymatic condition or centrifugation — in order to isolate the ASCs2,3. This method illustrates a simple procedure for isolating ASCs using an explant method, in the absence of harsh enzymatic or centrifugation steps.
The most common method of isolating ASCs consists of washing an adipose sample, enzymatically digesting the sample with collagenase, centrifuging the sample, and finally lysing the red blood cells prior to culturing the ASCs4. While efficient at isolating a high yield of ASCs, the use of xenogeneic components (e.g., enzymatic digestion with collagenase) is considered more than "minimally manipulated" by the U.S. Food and Drug Administration, and may pose risks such as immune reaction, prohibiting the cells' use in the clinic5,6. To minimize the risks of xenogeneic components, many groups have suggested non-animal derived, manufactured enzymes to digest the adipose tissue. However, these enzymes are still harsh, and can alter the cell phenotype7.
Other methods of isolating ASCs include high-speed centrifugation, using forces as high as 1,200 x g, and vortexing to isolate the ASCs8. Even forces as low as 400 x g were sufficient in isolating viable ASCs9. While these cells do produce a large quantity of viable cells, many protocols failed to proliferate beyond 14 days8. Further, mechanical isolation yields fewer recovered cells than enzymatic digestion, but a higher proportion of isolated cells were ASCs as compared to other cells endogenous to adipose tissue at passage 010.
The isolation of a purer, more viable population of ASCs in less time, coupled with the cost and risks of xenogeneic components, makes enzymatic digestion less and less appealing for translation to the clinic. While mechanical isolation is initially a favorable approach, there is significant disparity in the methods used, and the volume of tissue processed is limited to the size of the specialized centrifugal units, and can be dependent on operator consistency2.
While both enzymatic digestion and centrifugation quickly yield a high volume of ASCs, these isolated cells show phenotypic changes, yielding questions about their behavior when returned to a patient2. An explant-based method of ASC isolation, as described in this protocol, is thus being employed by some groups, whereby the ASCs migrate out of small pieces of solid adipose tissue3,11,12. This migration is likely an effect of the cells being drawn to the nutrient rich media. Like other populations of MSCs, ASCs adhere to plastic, and survive and proliferate in the used tissue culture media (components below), permitting their isolation from the other cell types from the adipose tissue. While fewer cells are initially recovered — often taking > 1 week until cells are visible on the tissue culture plate — these unmanipulated ASCs will proliferate in vitro, permitting expansion of the cells to clinically relevant volumes11,12,13.
The use of lipoaspirate and abdominoplasty samples has been approved by The Institutional Review Board (IRB) of Rutgers University — Newark Campus.
1. Preparation of Tissue Culture Media
2. Isolation of ASCs from Adipose Tissue
3 Culture of ASCs
4 Cryopreservation of ASCs
Using the method detailed here, ASCs were successfully isolated from lipoaspirate and abdominoplasty samples (Figure 1). After three passages, the isolated cells were found to have an MSC morphology (asymmetrical, spindle shape) and phenotype, similar to ASCs following enzymatic digestion (Figure 2). Prior studies from our group and others have shown these ASCs to differentiate into adipocytes, osteocytes, and neurons like other MSCs, including ASCs isolated by other means11,21.
In most cases, ASCs migration onto the tissue culture plate can be visualized within 3–5 days by bright field microscopy. However, in some cases, only sparse cells will be visible after 7 days. In rare cases, the cells will not migrate onto the plate and/or will not proliferate up until the third passage. This outcome generally correlates with a delay in processing the tissue sample.
Figure 1: Schematic representation of ASC isolation from abdominoplasty and lipoaspirate samples. Abdominoplasty and lipoaspirate samples were obtained as discard tissue following routine surgical procedures. Abdominoplasty samples were shredded, after which either abdominoplasty or lipoaspirate samples were plated as explants in tissue culture media. The ASCs migrated out of the tissue, towards the nutrient rich media. After 1 week, the explants were removed and ASCs cultured for downstream experimentation and/or clinical application. Please click here to view a larger version of this figure.
Figure 2: Isolated ASCs with an MSC morphology and phenotype. ASCs isolated by the enzyme-free, explant method have an MSC morphology and phenotype at passage 3. (A) Like other MSCs, these ASCs have an asymmetrical, spindle shape, whether isolated by explant or enzymatic method (e.g., collagenase). The ASCs isolated using the enzyme method yield a longer, narrower morphology as compared to the explant isolated ASCs; the latter closer resemble MSCs derived from other enzyme-free isolation methods, such as bone marrow derived-MSCs. (B) Like other MSCs, the explant isolated ASCs are negative for CD45 and positive for CD73, CD90, and CD105. Please click here to view a larger version of this figure.
ASCs are an attractive source of MSCs due to the easy access of the tissue. For both clinical and research applications, scientists must bear in mind donor variability when isolating and culturing these cells. For reasons yet to be elucidated, MSCs from different donors show different capacities to proliferate in vitro, which will subsequently affect the ASC's migration out of the adipose explant and proliferation capacity. While variability can be observed in the time it takes for the ASCs isolated from these enzyme-free explants to reach confluency, it was rare for a sample not to proliferate in vitro.
Beyond donor variability, the most likely source of failure of the cells to migrate out of the tissue and/or proliferate in vitro is the length of time that the tissue was stored prior to processing. When the samples were allowed to sit for >12 h prior to processing or were not kept moist between harvesting and processing, poorer migration and proliferation rates were observed. The only samples that frequently failed to proliferate were abdominoplasty samples that were not kept moist with a saline solution: in these cases, the tissue in the center of the tissue block was often moist enough to process, but occasionally needed to be discarded. It is thus critical to keep the tissue moist from time of harvest through processing.
In cases where ASCs are not observed on the plate at the 1 week mark, the adipose explants should still be removed from the tissue culture plates lest tissue necrosis occur. Sometimes there are too few cells to easily identify, but those few cells will be capable of expanding within the culture system. If the cells are still not observed at 3 weeks, the isolation should be considered failed.
In some cases, particularly of abdominoplasty, it is not possible to obtain a truly aseptic sample due to the limitations within the surgical arena. If it is not possible to use a sterile vessel to transport the tissue from the operating room to a laminar flow hood, removing the outer 1 cm of the tissue is generally sufficient to prevent microorganism contamination. If the abdominoplasty sample is attached to skin, the skin can be wiped with 70% ethanol to sterilize that surface prior to mincing the adipose tissue.
During the mincing process, it is critical that the tissue is minced into small, fine pieces. If the explants are too large, there will be insufficient surface area for the ASCs to migrate out of the tissue and onto the plate. Further, it is critical that the tissue does not remain in the plate any longer than necessary lest the tissue become necrotic.
A limitation of this method is the use of an enzyme (in the described protocol, trypsin) to detach the ASCs during the tissue culture process. Other non-animal derived enzymes, such as Accutase, can be replaced to remove the need for animal derived products, however, this will increase the cost of the tissue culture. For this reason, among others, numerous groups are investigating non-two-dimensional tissue culture methods such as bioreactors and scaffold-based systems to increase MSC growth potential while minimizing the need to detach the cells from their substrate22.
When moving ASCs to the clinic, a major limitation of the explant isolation method is the reliance on a good manufacturing practice (GMP) level tissue culture facility, which many medical centers lack. In these cases, any of the self-enclosed commercially available enzyme- or centrifugation-based methods would need to be employed. However, as GMP facilities become more prevalent, this effect is expected to be limited. In the meantime, even such facilities lacking GMP tissue culture facilities can consider using the explant method for studying ASCs in vitro: the less manipulation, the more akin these cells are to their in vivo counterparts11.
This method provides a simple way of isolating ASCs from adipose tissue — both lipoaspirates and abdominoplasties — in the absence of harsh enzymes or centrifugation steps. While the initial yield of ASCs is lower than that of other methods, the ASCs will proliferate in vitro, minimizing the effect of the lower initial yield. The lack of excessive or forceful manipulation makes ASCs isolated in such a manner particularly relevant, since there are fewer questions (i.e., whether the observed effects are due to the cells themselves or to the cells' manipulation during the isolation process).
The authors have no acknowledgements
Name | Company | Catalog Number | Comments |
Dimethyl Sulfoxide | Fisher Bioreagents | BP231 | |
Dulbecco's Modified Eagle's Medium - high glucose | Sigma-Aldrich | D5671 | |
Falcon 3003 tissue culture plates | Corning | Corning | |
Fetal Bovine Serum | Sigma-Aldrich | F2442 | serum is batch tested to ensure it supports MSC growth |
L-Glutamine solution | Sigma-Aldrich | G7513 | |
Mr. Frosty | Nalgene | 5100-0001 | |
Penicillin-Streptomycin | Sigma-Aldrich | P4333 | |
Trypsin-EDTA solution | Sigma-Aldrich | T4049 |
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