We conclude that the cell culture protocol presented in this note allows apparently complete differentiation of. Despite this, many believe organ culture to offer substantial benefits over adipocyte culture. Although murine adipose cell lines are useful for many mechanistic studies, primary. After 2 hours samples were covered with cell culture medium and cells were cultured. International orders are usually received in 34 days. Differentiation and characterization in primary culture of white adipose tissue brown adipocytelike cells article pdf available in international journal of obesity 2005 336. Due to their potential to differentiate from fibroblasts to adipocytes, 3t3l1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. That is a critical addition to both the adipocyte incubation solution and media. Replenish culture media every 23 days by aspirating using serological pipettes see note 15. Protocol for effective differentiation of 3t3l1 cells to. Taken together, these reports suggest that the inhibitory effects of these growth factors may depend on the origin, the state of development of the target preadipocytes, and culture conditions.
Atcc test for adipocyte formation for 3t3l1 cells atcc. Preparation of media preparation of media must be carried out in a tissue culture hood under aseptic conditions. Atmscs cultured in mesencultxf exhibit robust multilineage differentiation potential passage 2 atmscs readily differentiated into osteogenic a and adipogenic b lineages. Cells are shipped 5 days after initiating differentiation.
However, the validity of this method for quantitative analysis has not yet been established. Recent advances in bioengineering and cell biology of adipose tissue have led to. Characterization of immortalized human brown and white pre. Inoculate at 10,000 cells per cm 2 for rapid growth, or at 6,000 cells per cm 2 for regular subculturing. The exclusion of sera or all unidentified components from cell culture media is of high relevance for all tissue engineering trials. Understanding adipocyte differentiation physiological. The adipocyte precursor cells preadipocytes are isolated from. Cell chemotaxis was among the most enriched pathways p 3. Recent advances in bioengineering and cell biology of adipose tissue have led to new therapeutic potentials for regenerative medicine. Rapamycin inhibits human adipocyte differentiation in. The osteogenic cultures were stained with alizarin red to detect. Cai media are carefully fromulated and quality tested to confirm no bacteria, yeast or fungi contamination, and qc tests for correct ph, osmolality and lack of endotoxins.
Differentiation and characterization in primary culture of. The brown and white preadipocyte cell models terthba and terthwa, respectively displayed a stable proliferation rate and differentiation until at least passage 20. If an adipocyte phenotype is desired the preadipocytes must be differentiated into adipocytes, a process that takes two weeks following the. Second edition, several updated chapters are combined with many new contributions from internationally recognized experts covering both conventional and stateoftheart methodologies as well as whole animal, cellular and molecular approaches which have emerged in or been extended to adipose tissue research over the. Analysis of cell cycle arrest in adipocyte differentiation. Whether to use adipose tissue or adipocytes in your experiments is a decision which should not be taken lightly. Adipocyte cell culture is an important tool for mechanistic studies of energy metabolism.
We focus on those genetic events that link effectors to induction of adipocyte gene expression. This protocol can be used for human or rodent adscspreadipocytes. Second, coating cell culture dishes with collagen type i did not improve cell attachment to the dishes, whereas cell differentiation efficiency was comparable. Many factors affect the differentiation of adipocytes in culture. Identification of metabolically distinct adipocyte. Standard isolation of primary adipose cells from mouse. A simple culture method of fat cells from mature fat tissue fragments. Oil red o staining can be used to assess the degree of differentiation. Count the cells with a hemocytometer or cell counter.
Im starting the cell culture again using earlier passage, and im still waiting. It is the preferred method when assessing the longterm regulation of. Effect of rapamycin on human adipocyte differentiation. Using a 3d culture system to differentiate visceral. Isolation and culture of human mature adipocytes using. Protocol 3t3l1 preadipocyte differentiation agilent. Mature had are expected 10 days after induction of differentiation and should remain healthy and responsive for at least 2 weeks after complete differentiation. The cytosolic protein content of our primary cell cultures of preadipocytes in control medium 8. Using a cut ul pipet tip, very carefully remove the floating adipocyte layer to a fresh tube with warm krbh if performing further experiments or sdslysis buffer aspirate remaining liquid, being careful not to disrupt the pellet. Remove the 3t3l1 differentiation medium and replace with 3t3l1 adipocyte maintenance medium. Terminally differentiated cells are histochemically stained to determine their respective identities see below for staining protocol. This removes adipose tissue metabolites and secretory products free fatty acids, glycerol and cytokines, etc.
Adipose tissue plays a central role in the development of insulin resistance and. Adipogenesis has been extensively studied using in vitro model systems consisting either of established adipogenic cell lines clonal lines 3t3l1, 3t3f442a, ob17, bfc1, st, a31t. Cell culture basics handbook thermo fisher scientific. To determine if significant proliferation had occurred in our culture system, we measured cellular protein and dna synthesis. Chondrogenic differentiation and analysis of msc mesenchymal stem cell myocyte adipocyte osteoblast neuron chondrocyte selfrenewal perivascular progenitor cell osotetobblalstt chondrocyte. Here we provide a detailed protocol for the isolation of mature adipocytes from human adipose tissue and how to set up the maac system. In a very recent work, serumfree adipocytehuman umbilical cord vein ecs coculture was addressed at.
Patchclamp adipocyte isolation culture differentiation wholecell patchclamp perforatedpatch. If cells are to be maintained as preadipocytes, they should be fed with preadipocyte medium pm1 every other day. Preadipocytes are flat, phasedark spindleshaped cells. An sirnabased method for efficient silencing of gene expression in mature. Mature human adipocytes had are differentiated from hpad human preadipocytes through the use of adipocyte differentiation medium. Ecmadipocyte culture provides a physiologic environment that approximates the in vivo adipose tissue environment and provides a sustainable environment for preadipocyte stem cell growth and differentiation, as evidenced by upregulation of adipogenic genes in 3decmadipocyte culture. Adipose tissue at function is regulated by both shortterm and longterm mechanisms. This protocol describes primary in vitro culture of stromal cell isolated from either large or small quantities of human adipose tissue. The c3h10t1 2 cell line was established in 1973 from 14 to 17dayold c3h mouse embryos 15. Most chemicals are purchased from sigmaaldrich st louis, mo, cell culture. Optimal protocol for the differentiation and metabolic analysis of. Other cells, especially macrophages can adhere to isolated fat cells. Adipocyte media browse our specialized media, optimized for the unique requirements of adipocytes. Centrifuge at 500g 2200 rpm for 10 minutes to separate floating adipocytes from pelleted svf pre adipocytes and leukocytes.
Thus, primary adipocytes fresh or cultured are useful for acute metabolic studies. What is the efficient protocol for adipocyte differentiation of 3t3l1. Human adipocytes had fat cells from adipose tissue. This methodology permits the study of longterm adipocyte phenotypic changes and facilitates coculture of adipocytes with other cell types. All us and canada orders are shipped via federal express priority service and are usually received the next day.
Culture adiposederived mesenchymal stem cells in serumfree, xenofree mesencultxf figure 4. Several preadipocyte and stem cell culture models have been developed to improve the quality of tissueengineered fat by cultureexpanded adipocytes. The stroma vascular fractions of mouse bat and wat grown in primary culture in a brown adipocyte differentiation medium were studied. Pdf cultures of adipose precursor cells from brown adipose. Inhibiting methylation with 5azacytidine in c3h10t1 2 cells produces cells that exhibit stable. The cells have a similar appearance in culture to fibroblasts or smooth muscle cells see figure 1a. This protocol outlines how to chemically induce the differentiation of 3t3l1 cells into adipocytelike cells. Functional assessment of white and brown adipocyte. Primary cell culture and adipocyte differentiation. The 3t3l1as may be kept in a preadipocyte state by following a standard splitting and feeding protocol. All media used were supplemented with 100 uml penicillin, 0. Feed cells every 23 days using 3t3l1 adipocyte maintenance medium until ready for assay.
Protocol for effective differentiation of 3t3l1 cells to adipocytes. Primary cell culture is increasingly being used as a major tool in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. These cells display fibroblastic morphology in cell culture and are functionally similar to mesenchymal stem cells. Mature human white adipocytes cultured under membranes.
Longterm adaptations to hormonal, nutritional, and developmental factors take place over a. At 7080% confluency, trypsinize cells following steps 16 described in the. Gently aspirate the floating fat layer above the cells if visible. These apcs have distinct molecular phenotypes yet retain similar adipogenic potential. The highestrated network showed the functions cell chemotaxis, metastasis, formation of sprouting and protrusion, and filopodia figure 1f. Culture of isolated human adipocytes and isolated adipose tissue. Mature terthba adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature terthwa adipocytes. Place approximately 5,000 cellscm2 in tissue culture treated cultureware using 3t3l1 preadipocyte medium pm1l1.
Commitment of c3h10t12 pluripotent stem cells to the. While historically in the literature adiposederived stromal cells have been termed preadipocytes 14, 15, there is a growing appreciation that they are multipotent, with chondrogenic, neurogenic. The apcs give rise to adipocytes with divergent metabolic and endocrine capacities and their distribution varies in type 2 diabetes patients. Here we show that a protocol with arbitrarily chosen. Resuspend the cells in 5 ml of human preadipocyte growth medium by gently pipetting the cells to break up the clumps. Differentiation of 3t3l1 cells into adipocytelike cells. Robust and wellcharacterized, 3t3l1 pre adipocytes differentiate and form fat pads indistinguishable from normal adipose. Our protocol includes allowing the cells to reach confluence and then observing for the formation of lipid droplets over approximately 2 weeks. A novel growth factordependent thermogenic brown adipocyte cell line from. Quantitative assessment of adipocyte differentiation in. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available american type culture collection atcc 3t3l1 cell. We have cultured these unilocular fat cells by novel methods, namely, by the ceiling culture method 17, 18 and by.
In contrast to the upper body fat cell response, the number of lowerbody adipocytes did significantly increase during the course of experiment. We additionally show that these visceral adipocytes are functional fat cells that retain characteristics specific to the visceral depot. Human preadipocytes can be expanded in an undifferentiated state for future differentiation to mature adipocytes and show higher efficiency of adipogenesis. In this note, we present a detailed procedure for highly effective and reproducible 3t3l1 cell differentiation. Quantitative assessment of adipocyte differentiation in cell culture. This takes advantage of the floating adipocyte by allowing it to attach to the top of the tissue culture flask by filling the flask with media. Isolation and patchclamp of primary adipocytes springerlink. A human 3d extracellular matrixadipocyte culture model. This userfriendly 3d culture protocol will enable in vitro studies of primary visceral preadipocytes, which may lead to new mechanistic insights into the development and function of visceral fat. Robust and wellcharacterized, 3t3l1 preadipocytes differentiate and form fat pads indistinguishable from normal adipose. I tried to use trypan blue and hemocytometer but this way doesnt work cause cells are very big, always floating and can be disrupted easily. The differentiation efficiency varies depending on the donor. This article will discuss the various models used for studying the preadipocyte differentiation process, with the mouse 3t3l1 cell culture line described in detail.
Cells should completely disintegrate into individual cells, extend the time if necessary. Culture of isolated human adipocytes and isolated adipose. Adipocyte differentiation protocol adipocyte differentiation protocol is designed to achieve maximum adipocytes differentiation from preadipocytes or adiposederived stem cells adscs in vitro. Importantly, using this adipocyte culture method, we provide. In the upper body, an increase of adipocyte size correlated with upperbody fat gain. The answer is yes you can culture primary adipocytes in ceiling cultures. Human preadipocytes hpad culture protocol sigmaaldrich.608 1625 1372 587 1123 24 1532 972 712 1364 1279 990 1094 854 1200 1540 1647 771 1668 1065 786 973 553 99 278 1115 806 1359 453 714 1435 297 736 33 520 337 115 183 1387 456