Name: isolation of preadipocytes from broiler chick embryos
Uploaded: 2022-08-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Preadipocytes from Broiler Chick Embryos at JoVE.com

The authors thank UT AgResearch and the Department of Animal Science for supporting and optimizing this protocol. This work was funded by USDA grant.

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 &#176;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
<ol>
	<li>Prepare the culture hood and the instrument...

<ol>
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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Pipette</td>
			<td>Eppendorf</td>
			<td>Z683825</td>
			<td>Single Channel Pipette, 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>1 mL Pipette Tip</td>
			<td>Fisher Scientific</td>
			<td>02-707-402</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A426P4</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Flask</td>
			<td>Pyrex</td>
			<td>4980-25</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F75P-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>96-Well Assay Plate</td>
			<td>Costar</td>
			<td>3632</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Plate, Black Bottom</td>
			<td>Costar</td>
			<td>3603</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>AdipoRed</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench Top Wiper (Kimtechwiper)</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Up &#38; Up</td>
			<td>NDC 1167300334</td>
			<td>20% Working Solution</td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Corning</td>
			<td>6749</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer, 40 &#181;m</td>
			<td>SPL</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugaton</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken Serum</td>
			<td>Gibco</td>
			<td>16110082</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes, 15 mL</td>
			<td>VWR</td>
			<td>10025-690</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>Nunc</td>
			<td>343958</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps, 100 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-5137</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Surgical Scissors, 115 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-6839</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Millipore</td>
			<td>SYNSV0000</td>
			<td>Despensed as needed</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>HyClone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td>70% Working Solution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Evos</td>
			<td>M7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Plate Reader</td>
			<td>Biotek</td>
			<td>Synergy H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Foil</td>
			<td>Reynolds</td>
			<td></td>
			<td>Reynolds Wrap Heavy Duty Aluminum Foil, 125 SQ. FT.</td>
		</tr>
		<tr>
			<td>Freezing Container</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Millipore</td>
			<td>4055</td>
			<td>2% Working Solution</td>
		</tr>
		<tr>
			<td>Hematocytometer (Counting Chamber)</td>
			<td>Corning</td>
			<td>480200</td>
			<td>0.1 mm deep</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisher Scientific</td>
			<td>6845</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument Sterilizer</td>
			<td>VWR</td>
			<td>B1205</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic Acid-Oleic Acid-Albumin</td>
			<td>Sigma</td>
			<td>L9655</td>
			<td>1x Working Solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Evos</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Channel Pipette</td>
			<td>Thermo Scientific</td>
			<td>4661070</td>
			<td>12-Channel Pipetters, 30 - 300 &#181;L</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S-7907</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue</td>
			<td>Invitrogen</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma</td>
			<td>O-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>IKA</td>
			<td>KS130BS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Towel</td>
			<td>Tork</td>
			<td>RK8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm M</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Steptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>1x Working Solution</td>
		</tr>
		<tr>
			<td>Petri dishes, 100 mm</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 60 mm</td>
			<td>Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Shaker</td>
			<td>VWR</td>
			<td>200</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer</td>
			<td>Roche</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservior</td>
			<td>VWR</td>
			<td>89094-680</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Beaker, 100 mL</td>
			<td>Pyrex</td>
			<td>1000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Plate Reader</td>
			<td>Biotek</td>
			<td>Synergy H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>McKesson</td>
			<td>762703</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Forceps, 120 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-4960</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Scissors, 140 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-6762</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 Flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Strainer, 250 &#181;m</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td>0.1% Working Solution</td>
		</tr>
		<tr>
			<td>Tweezers, 110 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-5035</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 Collagenase</td>
			<td>Gibco</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 Filter Paper</td>
			<td>Whatman</td>
			<td>1001-110</td>
			<td></td>
		</tr>
	</tbody>
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isolation of preadipocytes from broiler chick embryos

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.

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 ...

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Isolation of Preadipocytes from Broiler Chick Embryos

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. ...

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipocyte development. This method has been optimized to yield cells with high viability and increased capacity to differentiate into mature adipocyte, which can be used for functional analysis of early life fat growth and development embryo in chicks.The process of adipogenic differentiation is highly comparable between chickens and humans. Therefore, isolated preadipocytes can be used as a dual-proposed model for studies relevant to humans and poultry. To begin swab the embryo body with 70%ethanol.Then to prevent interference during filtration at later steps after digestion gently scrubbed the skin surface with sterile gauze to remove feathers. Then cut off the skin between the legs and abdominal region to reveal the pair of femoral adipose depots. Use curved forceps with one hand to hold the skin around the leg and gently remove the femoral subcutaneous fat.with the other hand. Later, use curved forceps to gently pull the depot away from the leg. Transfer the tissues into a 15 milliliter tube containing five milliliters of collection media and repeat the dissection for the other side of the fat pad.Now, transfer the collected fat pads to a 60 millimeter Petri dish containing sterilization solution and rinse briefly by swirling the dish a few times. Then rinse the sterilization solution by transferring the fat pads to a 60 millimeter Petri dish containing PBS. Gently swirl the plate and once again, wash with PBS.For digestion of the adipose tissues, transfer them to a 15 millimeter tube containing pre-warm enzymatic solution. Then immerse a pair of long straight scissors in the tube and finally mince the adipose tissues in the solution into pieces as small as possible. Transfer the minced tissue and enzymatic solution to a 25 milliliter autoclaved flask.Wrap the flask with paraffin film and place the flask in an orbital shaker incubator at 37 degrees Celsius for digestion. After digestion, gently pipette up and down to mix well. Then remove any bits of undigested tissue and debris by filtering through a 250 micrometer tissue strainer into a 15 milliliter tube by pipetting.Now, remove the cells adhered to the flask by rinsing the flask with four milliliters of growth media and filter the cell suspension into the same 15 milliliter tube. Next rinse the strainer by pipetting the growth media again to remove any cells trapped in the filter. Centrifuge at 300 times G for five minutes at room temperature to pellet the cell fraction and aspirate the supernatant without disturbing the cell pellet.Gently resuspend the pellet in one milliliter of red blood cell lysis buffer, mix well by pipetting and incubate for five minutes at room temperature. Then dilute the cells by adding five milliliters of growth media to the tube containing the cells and mix them gently by pipetting. Now, pipette the cells onto a 40 micrometer cell strainer and filter them into a new 50 milliliter tube.Then rinse the strainer with an additional five milliliters of growth media and pellet the cells by centrifuging at 300 times G for seven minutes at room temperature. Carefully aspirate the supernatant. Resuspend the remaining cell pellet in one milliliter of growth media by pipetting To determine the cell density and viability, mix 10 microliters of each sample in trypan blue by pipetting.Load 10 microliters of the mixture onto the hemocytometer and count the cells. Analysis of preadipocytes after 24, 48, and 72 hours showed normal cell morphology and number. Adipogenic induction of preadipocytes showed rapid differentiation and accumulation of lipid droplets.Aggressive handling of preadipocytes during isolation resulted in cell damage and low cell number. Microbial contamination can be observed despite taking preventative measures. Lipid staining with oil red O indicated the preadipocytes quickly begin to develop lipid droplets under adipogenic inducement and accumulation progresses over time as observed in 24, 48, and 72 hours.The lipid accumulation relative to cell number was quantified using lipid and DNA stains. Both the lipid accumulation and cell proliferation increased over time. Low cell number or damaged cells can be observed when the tissue fractions are digested too long.Therefore, it is recommended that one and a half hour of digestion is not exceeded. Isolated preadipocytes can be used for gene expression studies. Sufficient RNA can be obtained for use in cDNA synthesis and follow-on qPCR or RNAseq.

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