The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.
Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.
Obesity is a global health threat to both adults and children. Children who are overweight or obese are approximately five times more likely to be obese as adults, placing them at significantly increased risk for cardiovascular disease, diabetes, and many other comorbidities. About 13.4% of US children aged 2-5 have obesity1, illustrating that the tendency to accumulate excess body fat can be set in motion very early in life. For very different reasons, the accumulation of excess adipose tissue is a concern for broiler (meat-type) chickens. Modern broilers are incredibly efficient but still accumulate more lipid than is physiologically necessary2,3. This tendency begins soon after hatch and effectively wastes feed, the most expensive production component, by allocating it away from muscle growth. Therefore, for both children and broiler chickens, albeit for very different reasons, there is a need to understand factors that influence adipose tissue development and identify ways to limit adipose expansion early in life.
Adipocytes form from preadipocytes, adipose tissue-derived stem cells that undergo differentiation to develop mature, lipid-storing fat cells. Accordingly, preadipocytes in vitro are a valuable experimental model for obesity studies. These cells, isolated from the stromal vascular fraction of adipose depots, can provide a fundamental understanding of molecular pathways controlling adipocyte differentiation and metabolism4,5. Chick embryos are a favorable experimental model in developmental studies because culturing eggs on the desired schedule makes experimental manipulation easier, as it enables obtaining embryos without the mother’s sacrifice to observe a series of developmental stages of embryos. Moreover, complicated surgical procedures and lengthy periods of time are not required to obtain embryos relative to larger animal models. Therefore, the chick embryo presents an opportunity to obtain preadipocytes from the earliest stages of adipose tissue development. Subcutaneous adipose tissue becomes visible in the chick around embryonic day 12 (E12) as a clearly defined depot located around the thigh. This depot is enriched in highly proliferative preadipocytes that actively undergo differentiation under developmental cues to form mature adipocytes6,7. The process of adipogenic differentiation is comparable between chickens and humans. Therefore, preadipocytes isolated from chick embryos can be used as a dual-purpose model for studies relevant to humans and poultry. However, the yield of preadipocytes declines with aging as cells grows into mature adipocytes5.
The present protocol optimizes the isolation of preadipocytes from adipose tissue during the stage (E16-E18) at which adipogenic differentiation and adipocyte hypertrophy are at their peak in broiler chick embryos8. This procedure can assess the effects of factors to which the developing embryo is exposed in ovo, such as the hen diet, on adipocyte development and adipogenic potential ex vivo. It can also test the impact of various manipulations (e.g., hypoxia, nutrient additions, pharmacological agonists, and antagonists) on adipogenesis or the various ‘omes (e.g., transcriptome, metabolome, methylome) of adipocyte progenitors. As a representation of the earliest stage of adipose formation, cells obtained using this protocol are valuable models for studies relevant to poultry and humans.
All animal procedures were approved by the University of Tennessee Institutional Animal Care and Use Committee. Freshly fertilized commercial broiler eggs (Cobb 500) were obtained from a local hatchery. Eggs were incubated at 38 °C with 60% relative humidity until dissections at embryonic days 16-18 (E16-E18). Adipose tissue was collected from the subcutaneous (femoral) depot.
1. Preparation for isolation and culture
2. Adipose tissue collection and digestion
3. Seeding and culture of preadipocytes
4. Subculturing and cryopreservation
5. Adipogenic differentiation
NOTE: 2% gelatin-coated plates can be used to enhance cell adhesion.
6. Assessing adipogenesis
Primary preadipocytes are morphologically similar to fibroblasts, with irregular, star-like shapes and a central nucleus (Figure 2A–C). The cells readily adhere to tissue culture plastic and begin to proliferate soon after attachment. They rapidly differentiate and accumulate lipid droplets (Figure 3D) when provided with fatty acids in the media. The viability (98%, based on dye exclusion) reported in the isolations represented here is typical. While the cells are fairly robust, aggressive handling during isolation leads to cell damage (Figure 3E), yielding cells that attach poorly and fail to proliferate. Despite the procedures incorporated to prevent microbial contamination, transfer of non-sterile material can occur (Figure 3F). Because of their rapid growth rate, chick embryo preadipocytes consume glucose in the media at high rates. Media should be changed every 48 h to maintain their supply of energy.
The representative results presented here illustrate the adipogenic potential of chick embryo preadipocytes. These cells quickly accumulate to develop lipid droplets under adipogenic conditions, and accumulation progresses over time (Figure 4 and Figure 5). Two methods have been presented which can be used to better visualize and quantify the degree of lipid accumulation in these cells, which is a direct reflection of adipogenesis. Staining lipids with Oil Red O is a low-cost method to visualize and quantify the accumulation of lipid droplets (Figure 4). A light microscope is used to collect images, and stained cells can be held on the benchtop until images are collected. Lipid accumulation in each dish of cells can be quantified as described by extracting the stain and reading the absorbance at 495 nm using a spectrophotometer. Using the combination of the selected lipid and DNA stains, it was possible to quantify the lipid accumulation relative to cell number, which compensates for non-adipogenic cells that may persist in culture (Figure 5). If measures are taken over several time points (Figure 5B-C), this combination makes it possible to assess both adipogenesis and proliferation, for example in response to added hormones or peptides.
Figure 1: Adipose tissue collection. (A) On breaking the eggshell, the white shell membrane was revealed. (B) Piercing the amnion using sterile tweezers. (C) A total of ~80 mg of femoral subcutaneous fat can be obtained from E16. Please click here to view a larger version of this figure.
Figure 2: Tissue fragments for enzymatic digestion and cell pellet after digestion. (A) Minced adipose tissue in enzymatic solution (~1 mm3). (B) Arrows indicate cell pellets after RBC lysis. Please click here to view a larger version of this figure.
Figure 3: Cell morphology comparisons of isolated primary preadipocytes. (A) Preadipocytes at 24 h after isolation. (B) Preadipocytes at 48 h after isolation. (C) Preadipocytes with 80% confluency at 72 h after isolation. (D) Preadipocytes after 48 h adipogenic induction at passage 4, lipid droplets are visible. Inset indicates the magnified image of lipid droplets. (E) Representative image of damaged cells. (F) Arrow indicates black swimming dots in contaminated culture. Scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 4: Evaluation of lipid accumulation by Oil Red O staining. (A) Representative images of Oil Red O staining of E16 preadipocytes after 24 h, 48 h, and 72 h adipogenic differentiation ex vivo. Scale bars = 100 µm. (B) Quantification of lipid droplets measured by elution of Oil Red O staining. Values are expressed as mean ± SD. a,b,c P < 0.05 by one-way ANOVA with posthoc Tukey's HSD test. Please click here to view a larger version of this figure.
Figure 5: Assessment of adipocyte differentiation by lipid stain/DNA stain. (A) Representative images of the lipid stain (red) and the DNA (blue) staining of E16 preadipocytes after 24 h, 48 h, and 72 h adipogenic differentiation ex vivo. Scale bars = 150 µm. (B) Lipid staining (Excitation: 485 nm/Emission: 572 nm) was performed to assess lipid accumulation in differentiated preadipocytes. (C) DNA staining (Excitation: 359 nm/Emission: 450 nm) was performed to assess variations in the number of cells. (D) The ratio of lipid and DNA stain. Lipid accumulation is normalized to the DNA content. Values are expressed as mean ± SD. a,b,c P < 0.05 by one-way ANOVA with posthoc Tukey's HSD test. Please click here to view a larger version of this figure.
Age (n=) | x106 cells/100 mg tissue | Viability (%) |
E12 (4) | 0.97 ± 0.115 a,b | 98.5 ± 0.58 a |
E14 (4) | 1.22 ± 0.232 a,b | 98.3 ± 0.96 a,b |
E16 (21) | 1.61 ± 1.717 a | 97.6 ± 1.58 a |
E17 (4) | 0.81 ± 0.282 a,b | 96.8 ± 2.63 a,b |
E18 (7) | 0.72 ± 0.611 a,b | 95.9 ± 1.81 a,b |
E20 (4) | 0.94 ± 0.171 a,b | 97.8 ± 0.8 a,b |
D4 (9) | 0.24 ± 0.164 a,b | 93.6 ± 4.28 b |
D5 (4) | 0.25 ± 0.073 a,b | 98.5 ± 0.71 a,b |
D7 (10) | 0.17 ± 0.162 b | 96.8 ± 3.49 a,b |
D14 (4) | 0.25 ± 0.051 a,b | 99.0 ± 0.00 a,b |
Table 1: Average cell number and viability of isolated cells from embryo and post-hatch chicks.
Values are expressed as mean ± SD. a,b P < 0.05 by one-way ANOVA with posthoc Tukey's HSD test.
Although several well-described protocols have reported the isolation of preadipocytes14,15,16,17, isolation for embryonic preadipocytes has been optimized, which can be used for functional analyses of early life fat growth and development in broiler chicks. This protocol yields high viability embryonic adipocyte progenitors with high differentiation potential. Moreover, the presented procedure for isolating preadipocytes is not limited to embryos but can be used in post-hatch chicks. However, it has been optimized for use with E16 embryos, and yields from chicks after hatch are considerably lower (Table 1), likely due to increases in the relative amount of connective tissue as chicks grow rapidly.
The success of cell isolation is ultimately evaluated by observing the shape and number of attached cells (Figure 3A). Low cell number or damaged cells can be observed when the tissue fractions are digested too long, especially when tissue dissociation progresses for more than 1.5 h (Figure 3E). Therefore, it is recommended that 1.5 h of digestion is not exceeded. On the other hand, if the tissue fragment remains clearly after an hour of digestion, the digestive time or the amount should be increased. The obtained adipose tissue amount also might be varied depending on the genetic strain of the chicken. If this protocol is used to isolate cells of other ages or species, both the amount of collagenase and digestion time will likely need to be modified.
A common limitation of primary cell culture is that isolated cells begin to lose adipogenic potential after several passages in culture; thus, it is important to induce differentiation within a few days of isolation to ensure the embryonic preadipocytes do not lose their adipogenic ability. Adipogenic precursors of embryos readily differentiate into mature adipocytes in the media formulation described above, without the need for confluence or hormonal induction. The cells up to passage 4 (10-14 days) are easily differentiated within 48 h of inducement (Figure 3D).
Controlling cell contamination in primary cell culture is another challenge, where fungal contamination is the most common. The use of Amphotericin B, as described, is generally effective at preventing fungal growth18,19. It can be included at low concentrations in the culture media with no noticeable effects on viability or adipogenic potential. Unidentifiable microbial contaminants have been observed in the form of black swimming dots appearing a few days after cell isolation (Figure 3F). These adversely affected cell growth and were impossible to remove once this infection occured20. Whether these contaminants were an unknown microbial species found in or on eggs or arose from another source is unknown. This protocol attempts to minimize the risk of contamination by extracting embryos aseptically from the egg21. Using a heat-based instrument sterilizer in the hood during dissection also helps to minimize the potential for contamination, especially when multiple embryos are dissected.
One consideration in the process is decreased cell adhesion to tissue culture plate during differentiation, resulting from physical changes as cells form and expand lipid droplets. Although contained within the cells, lipid droplets are buoyant and, when large, appear to act as balloons that tend to promote cells lifting from the surface. Thus, care should be taken in handling preadipocytes while inducing differentiation, and particularly when adipogenesis is assessed. While the protocol presented here does not modify the culture surface, cell adhesion can be enhanced when plating cells on dishes coated with 2% gelatin. It is also important to confirm the pH of wash solutions to be 7.4 and to use pre-warmed PBS solution when washing cells and mixing with the DNA stain to reduce cold stress.
Using multiple embryos, sufficient numbers of cells can be isolated to yield experimental replicates without the need for multiple passages, expansion and the risk of cells losing their adipogenic potential. RNA can be easily isolated from both pre- and differentiated chick embryo adipocytes isolated using phenol- or membrane-based commercial methods for gene expression studies. Sufficient RNA can be obtained from a single well of a six-well plate for use in cDNA synthesis and follow-on qPCR or RNAseq.
In summary, the accessibility of adipose depots in the chick embryo to isolate a cell model is highly relevant for both poultry and humans. This protocol is relatively simple to perform, and it yields a high percentage of viable cells that can be readily induced to undergo adipogenic differentiation in vitro. Fertilized chicken eggs can be obtained from various commercial sources for minimal cost, making these cells a readily available model for practical use.
The authors have nothing to disclose.
The authors thank UT AgResearch and the Department of Animal Science for supporting and optimizing this protocol. This work was funded by USDA grant.
1 mL Pipette | Eppendorf | Z683825 | Single Channel Pipette, 100 – 1000 µL |
1 mL Pipette Tip | Fisher Scientific | 02-707-402 | |
100% Isopropanol | Fisher Scientific | A426P4 | |
1x PBS | Gibco | 10010023 | |
25 mL Flask | Pyrex | 4980-25 | |
37% Formaldehyde | Fisher Scientific | F75P-1GAL | |
6-Well Plate | Falcon | 353046 | Tissue Culture-treated |
96-Well Assay Plate | Costar | 3632 | |
96-Well Plate, Black Bottom | Costar | 3603 | Tissue Culture-treated |
AdipoRed | Lonza | PT-7009 | |
Amphotericin B | Gibco | 15290026 | |
Bench Top Wiper (Kimtechwiper) | Kimberly-Clark | 34155 | |
Betadine | Up & Up | NDC 1167300334 | 20% Working Solution |
Cell Counter | Corning | 6749 | |
Cell Strainer, 40 µm | SPL | 93040 | |
Centrifugaton | Eppendorf | 5702 | |
Chicken Serum | Gibco | 16110082 | |
Conical Centrifuge Tubes, 15 mL | VWR | 10025-690 | |
Conical Centrifuge Tubes, 50 mL | Falcon | 352098 | |
Cryovial | Nunc | 343958 | |
Curved Forceps, 100 mm | Roboz Surgical | RS-5137 | |
Curved Surgical Scissors, 115 mm | Roboz Surgical | RS-6839 | |
Distilled Water | Millipore | SYNSV0000 | Despensed as needed |
DMEM/F12 | HyClone | SH30023.01 | |
DMSO | Sigma | D2650 | |
Ethanol | Decon Labs | 2701 | 70% Working Solution |
Fetal Bovine Serum (FBS) | Gibco | 10437028 | |
Fluorescent Microscope | Evos | M7000 | |
Fluorescent Plate Reader | Biotek | Synergy H1 | |
Foil | Reynolds | Reynolds Wrap Heavy Duty Aluminum Foil, 125 SQ. FT. | |
Freezing Container | Thermo Scientific | 5100-0001 | |
Gelatin | Millipore | 4055 | 2% Working Solution |
Hematocytometer (Counting Chamber) | Corning | 480200 | 0.1 mm deep |
Incubator | Fisher Scientific | 6845 | |
Instrument Sterilizer | VWR | B1205 | |
Linoleic Acid-Oleic Acid-Albumin | Sigma | L9655 | 1x Working Solution |
Microscope | Evos | AMEX1000 | |
Multi-Channel Pipette | Thermo Scientific | 4661070 | 12-Channel Pipetters, 30 – 300 µL |
Na2HPO4 | Sigma | S-7907 | |
NaH2PO4 | Sigma | S-3139 | |
NucBlue | Invitrogen | R37605 | |
Oil Red O | Sigma | O-0625 | |
Orbital Shaker | IKA | KS130BS1 | |
Paper Towel | Tork | RK8002 | |
Parafilm | Parafilm M | PM996 | |
Penicillin/Steptomycin (P/S) | Gibco | 15140122 | 1x Working Solution |
Petri dishes, 100 mm | Falcon | 351029 | |
Petri dishes, 60 mm | Falcon | 351007 | |
Plate Shaker | VWR | 200 | |
RBC Lysis Buffer | Roche | 11814389001 | |
Reagent Reservior | VWR | 89094-680 | |
Small Beaker, 100 mL | Pyrex | 1000-100 | |
Spectrophotometer Plate Reader | Biotek | Synergy H1 | |
Sterile Gauze | McKesson | 762703 | |
Straight Forceps, 120 mm | Roboz Surgical | RS-4960 | |
Straight Scissors, 140 mm | Roboz Surgical | RS-6762 | |
T-25 Flask | Corning | 430639 | Tissue Culture-treated |
Tissue Culture Incubator | Thermo Scientific | 50144906 | |
Tissue Strainer, 250 µm | Pierce | 87791 | |
Trypan Blue Stain | Gibco | 15250061 | |
Trypsin | Gibco | 15400054 | 0.1% Working Solution |
Tweezers, 110 mm | Roboz Surgical | RS-5035 | |
Type 1 Collagenase | Gibco | 17100017 | |
Water Bath | Fisher Scientific | 15-462-10 | |
Whatman Grade 1 Filter Paper | Whatman | 1001-110 |