Isolation of Mature Adipocytes and Stromal Vascular Cells under Adverse Sampling Conditions

Copyright: © 2012 Duarte MS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Adipocytes are cells that comprise a portion of any adipose depot. While they are not the only cells present in the adipose depot [1], discernible adipocytes function to assimilate moderate and long-chain fatty acids, store them as triacylglycerol, and release the fatty acids in times of negative energy balance, or in response to a repartitioning agent [1,2]. Adipocytes of some adipose depots also synthesize and release regulatory cytokines into the blood, which have been postulated to regulate a variety of whole animal and local non-adipose tissue physiology [1,3,4]. As such, in addition to moderating aspects of energy balance, adipocytes also may play a role in altering the body's blood pressure [3,4], ability to respond to insulin, control the rate of cellular apoptosis, fight inflammation and maintain normal levels of connective tissue surrounding tissues [5,6]. Adipocytes have been directly linked to obesity, metabolic syndrome, hypertrophy of the heart, and numerous other dynamic pathologies [7][8][9].
Stromal Vascular (SV) cells in adipose depots are present and active in the presence of fully differentiated adipocytes [10], and rodentderived cells have largely been used as a model for defining aspects of adipogenesis [1]. Are there other animal models that may help in defining aspects of adipocyte physiology, whereby adipocytes from all adipose depots are abundantly available? Research with large animals (such as cattle) provides such an animal model. These types of animals form the foundation of the National Institute of Health/United States Department of Agriculture directive towards use of "dual purpose animals" for both human and animal production-related research, because these large animals are good models/donors for studying molecular/cellular mechanisms of adipocyte physiology. For example, cattle are sufficiently large that all adipose depots may be easily sampled at the same time for comparative adipogenesis studies (depot vs depot), or individually (intramuscular depot) for adipogenesis and lipid metabolism studies [11,12]. In addition, the expanding marker databases of cattle promote the usage of this model.
Previous research with beef-derived adipocyte isolation utilized existing animal facilities in controlled environments such as either a local surgical suite or a meat laboratory (abattoir). This allows one to be capable of controlling the environment to which tissues are initially prepped/isolated/handled and packaged for transport. Moreover, tissue obtained for cell isolation could be extracted from the donor animal fast, efficiently transferred to a sterile (buffer) environment, and (all) promptly returned to the cell culture laboratory for processing. What about those without such facilities such as urban medical schools, researchers at (very) small colleges or other countries whereby facilities are quite limited? Can tissues be isolated from beef cattle if none of the environmentally controlled facilities are available? The focus of this paper was to obtain skeletal muscle from beef animals, which were being terminated and initially processed by a custom-packing/ processing company with on-the-farm service. If successful, such tissue procurement results may immediately suggest that research personnel with few facilities might be in a position to conduct research in this area.

Animals and pre-slaughter material preparation
Animals used for tissue sampling were slaughtered in a beef farm located in Viola, Idaho-USA by using a mobile slaughter unit ( Figure  1). No attempt was made to influence their normal procedures. Instead, representatives of the commercial entity physically provided samples to laboratory personnel. Immediately after receipt of the samples, laboratory individuals returned to the cell culture laboratory expediently. However, the locale and personnel responsible for handling of the live animals/slaughter of the same/obtaining the muscle samples, and providing the samples to the laboratory personnel was new and never used for any work like this previously. Thus, we were not able to control any of the steps in tissue isolation-other than to request the specific muscle that should be sampled. Cattle were simultaneously slaughtered by cerebral concussion followed by jugular venesection

Abstract
Methods are described to obtain adipose tissue for cell isolation, under adverse isolation conditions whereby no scientific controls were in place. Such methods could be used by laboratories of institutions where controlled environments (surgery rooms, abattoirs) are not available. While not ideal, we show that a variety of adipocytes and adipocyte-like cells may be isolated from such methods. These types of procedures may facilitate a greater number of persons entering into the research arena with adipocytes, and are easily adaptable to other animal models. and the muscle samples were collectively collected immediately after bleeding.
The cell culture laboratory was prepared for cell isolation procedure one day before the tissue sampling. All the supplies needed including plastic and glassware, and buffer solution (PBS) were sterilized prior the tissue collection in order to optimize the cell isolation procedure. The buffer solution was warmed in a water bath to 37°C and kept in a thermal box during the pre-slaughter until the samples collection in order to keep it as warm as possible.

Tissue samples
At the farm, samples of sternomandibularis muscle from four Angus cattle were collected within 10 min after slaughter and immediately placed in a sterile beaker containing warm phosphate buffered saline (PBS) supplemented with 100 IU/ml penicillin, 100 µg/ ml streptomycin, 2.5 ng/ml Fungizone B and 50 µg/ml Gentamicin. The beakers containing the tissue were placed in a thermal box as soon as the samples were collected in order to keep the samples warm, and then taken to the cell culture laboratory. As soon as the samples arrived at the cell culture laboratory they were immediately processed 50 min after collection and delays were avoided on the way back to the cell culture laboratory in order to short the time between sampling and cell isolation. A timeline of the sample collection and cell isolation procedure is presented in Figure 2.

Cell isolation procedure
Mature Adipocytes (MAs) and Stromal Vascular (SV) cells from Intramuscular Fat (IMF) depot were isolated as described by Fernyhough et al. [13] with minor modifications as it follows ( Figure  3). Preparation of reagents and media used for cell isolation is described in Table 1. 1. Rinse the muscle sample with PBS (37°C, pH = 7.08) supplemented with antibiotics/antimycotics and trim it prior to muscle dissection in order to reduce chances of contamination.
2. In a laminar flow hood, place the sample in a sterile dish, bath in PBS. Using sterile forceps and scissor dissect the muscle tissue in order to access the intramuscular fat depots. Once beginning the sample dissection, do not turn the tissue upside down since to keep the interior of the sample as clean as possible.

Photomicrographs
All photomicrographs were taken with a Sony RGB digital camera (3/4-inch chip) coupled to a Nikon Diaphot phase contrast microscope and Image Pro Plus ® image analysis software. The major equipment,

Results and Discussion
It is fairly clear that the environment in which tissue culture is carried out must be clean. This concept leads us to think that for a successful isolation procedure tissue handling must be done with extremely aseptic manner by trained individuals in order to avoid any source of contamination and transported to cell culture laboratory as soon as possible. As such, to isolate cells from a tissue in order to obtain primary cell culture more than an equipped cell culture laboratory is required, being necessary to be as close as possible to the place that the tissues are obtained. Additionally, one of the most important considerations that should be taken prior to cell isolation is the cooperation and collaboration of the clinical staff, which can be easily achieved if a person responsible for tissue isolation is also a member of the cell culture laboratory.
From the moment that a biological sample is removed from its natural environment within the animal, it is susceptible to deterioration from external factors such as moisture loss and temperature fluctuation and excessive passage of time. Another threat to the success of any cell isolation method is contamination from microbial sources including the host's own natural flora. All phases of the isolation process offer opportunities for contamination to occur unless strict adherence to proper aseptic techniques is consistently followed [14]. Additionally, the availability of the cells isolated from a tissue sample also depends on how fast the tissue sampling, dissection and digesting can be done, as in most cases contamination problems are related to the time [15].
In this study Mature Adipocytes (MA; Figure 4) and Stromal Vascular (SV; Figure 5) cells were successfully isolated from bovine muscle collected under non-asepsis conditions. No signs of contamination were detected by microscopy visualization such as cloudiness of media, deterioration of the cells and detachment of the cell monolayer, which would indicate mycoplasm contamination [16,17]. The photomicrographs results clearly shows that a great number of cells were successful isolated even though the tissue was collected under adverse conditions and was completely exposed to a non-aseptic environment prior to cell isolation procedures, which was never done in this laboratory before. Additionally, due to the distance between the local of tissue sampling and the cell culture laboratory, the samples were not promptly processed as suggested by several previously studies [14,18,19].
The results obtained in this study encourage new researchers in cell culture field by showing a protocol that allows the isolation of cells from muscle tissue without proper animal facilities. By using the cell isolation procedure developed in our laboratory additionally to the use of basic sterile techniques at the cell culture facility, we were able to obtain non-contaminated, healthy, primary cultures of cells. However, even though we were able to isolate cells in an adverse scenario, it is still recommended the use of sterile techniques during sample collection to avoid contaminations and cell death.