Name: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots (Video) | JoVE Journal
Uploaded: 2015-06-02T12:00:00.000+00:00
Duration: 8 min 30 s
Description: Learn encapsulation of thermogenic preadipocytes for transplantation into adipose tissue depots using alginate microbeads. This method enables enhanced energy dissipation and supports obesity research applications.

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5 minutes

We would like to thank Jennifer Petrosino and David DiSilvestro for editorial help. This research was supported by Award Number 20020728 from the American Egg Board and Award Number 10040042 from Novo Nordisk Pharmaceuticals as well as by the Food Innovation Center, Office for International Affairs, Center for Advanced Functional Foods Research, and Entrepreneurship at OSU as well as the National Science Foundation grant EEC-0914790 (L.J.L). The project described was supported by Award Number R21OD017244 (O.Z.) and UL1RR025755 (OSUCCC) from the National Center for Research Resources, funded by the Office of the Director, National Institutes of Health (OD) and supported by the NIH Roadmap for Medical Research and &#160;NCI P30CA16058. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.

The study protocol was approved by The Ohio State University Ethics Committees. Animal experiments were approved by IACUC protocol. All procedures were performed under the level 2 biosafety cabinet with laminar flow. We followed all standard safety requirements and procedures. The microencapsulation technique for preparation of microcapsules has been performed as described17,18.
1. Preparations of Materials
<ol>
	<li>Prepare 10 ml of 2% sodium alginate solution in autoclaved physiological saline (0.9% NaCl in water). Prepare 0.05% PLL solution in physiological saline. Prepare these solutions the day before and stir overnight. Filter the solutions with 0.22 &#956;m filter before use.</li>
	<li>Prepare 50 mM sodium citrate and 100 mM CaCl2 in 0.9% NaCl solutions and autoclave them.</li>
	<li>Autoclave all solutions, needles, electrodes, and beakers. Thoroughly clean needles with wires to avoid clogging.</li>
	<li>Determine the appropriate amount of cells to use for the capsules (102 microcapsules are needed for every cm2 of well and there are approximately 500 cells per capsule18). Use 1 ml of sodium alginate per two million cells.</li>
	<li>Prepare a &#8216;Fibroblast Growth Medium&#8217; containing 10% calf serum, and 100 U/ml penicillin/streptomycin in a high glucose (4,500 mg/l glucose) Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM).
	<ol>
		<li>Prepare a &#8216;Differentiation Medium I&#8217; containing 10% fetal bovine serum, 10 &#181;g/ml insulin, 1 &#181;M dexamethasone, 0.5 mM 3-isobutyl-1-methyl xanthine, and 100 U/ml penicillin/streptomycin in DMEM.</li>
		<li>Prepare a &#8216;Differentiation Medium II&#8217; containing 10% fetal bovine serum, 10 &#181;g/ml insulin, and 100 U/ml penicillin/streptomycin in DMEM.</li>
	</ol>
	</li>
	<li>Prepare lysis buffer containing one protease inhibitor tablet per 10 ml Radio-Immunoprecipitation Assay buffer (RIPA buffer).</li>
</ol>
2. Alginate Microbeads Preparation (Figure 1A)
<ol>
	<li>Remove old medium from cell culture flask. Rinse cells with 10 ml of PBS. Bring cells in suspension with 0.25% trypsin-EDTA (2 ml per one confluent T175 flask).</li>
	<li>Count cells in cell suspension using a hemocytometer. Use 10 ml aliquot from the cell suspension and count cells according to manufacturer&#8217;s instructions. Centrifuge the remaining cells in centrifugation medium at 480 x g at room temperature for 5 min.</li>
	<li>Suspend the cell pellet in sodium alginate as described in step 1.4. Transfer the sodium alginate-cell solution to a 5 ml syringe.</li>
	<li>Remove air bubbles in the solution, add a 23-gauge needle and invert the syringe to create a 1 ml pocket of air.</li>
	<li>Place a small beaker (180 ml) containing 144 ml of 100 mM CaCl2 solution under the needle spout of the encapsulator. Attach the electrode to the encapsulator with the tip approximately 2.5 cm above the surface of the 100 mM CaCl2 solution.</li>
	<li>Place tightly the syringe containing the sodium alginate-cell solution in the syringe pump. Attach the rubber tube to the opening of the syringe. Push the plunger until the sodium alginate-cell solution enters halfway through the tube. Adjust the voltage to 5.4 kV. Set the 12.06 mm diameter on the syringe pump. Adjust the speed to 3 ml/hr. Start the pump. Turn on the encapsulator and maintain the voltage at 5.4 kV.</li>
	<li>Close the window of the hood and avoid any unnecessary vibration till the end of formation of the alginate microbeads. After all solution passes through the needle, solidify the alginate ball-shaped microbeads in the 100 mM CaCl2 solution for additional 20 min before coating with PLL.</li>
</ol>
3. Coating Microbeads with PLL (Figure 1B)
<ol>
	<li>Remove the beaker containing the sodium alginate-cell ball-shaped microbeads and transfer these microbeads into a 50 ml centrifugation tube. Remove CaCl2 solution from the microbead pellet.</li>
	<li>Wash the alginate-cell ball-shaped microbeads by adding 30 ml of 0.9% NaCl. Shake the tube by hand gently. Remove 0.9% NaCl with 25 ml pipette after the microbeads have precipitated by gravitation. Repeat two more times for a total of 3 washes.</li>
	<li>Use 10 ml of 0.05% PLL solution for every 1 ml of sodium alginate solution. Add the 0.05% PLL and vortex at 1,000 rotations per min for 10 min. 
	Note: Usually, 10 min is sufficient for PLL coating.</li>
	<li>After PLL coat is formed, remove the PLL solution and wash the capsules 3 times as described in 3.2.</li>
</ol>
4. Removal of Alginate Core (Figure 1C)
<ol>
	<li>Add 30 ml of 50 mM sodium citrate solution. Wait 5 min or until all the sodium alginate is dissolved. Wash capsules three times as described in step 3.1.1.</li>
	<li>Remove the 0.9% NaCl, add 20 ml of culture medium to the 50 ml tube and transfer all capsules containing the cells to a cell culture flask. Handle encapsulated cells under standard cell culture conditions18.</li>
</ol>
5. In Vitro Applications to Study Xenograft and Host Cell Interactions or Kinetics of Metabolite Influx/Efflux between Cells (Figure 2)
<ol>
	<li>Culture host cells on 24 well plate until confluent for co-cultures. Use the &#8216;Fibroblast Growth Medium&#8217; for culturing preadipocytes.</li>
	<li>Transfer microcapsules into 24 well plate containing confluent host cells. Add microcapsules to achieve a monolayer (102 microcapsules/cm2 of well).</li>
	<li>Induce pre-adipocyte differentiation with Differentiation Medium I. Every 48 hr, change media to Differentiation Medium II &#160;for six days. Lyse cells in RIPA buffer. Use 50 &#181;g protein per condition to analyze protein expression using Western blot.</li>
</ol>
6. In Vivo Application for Treatment of Obesity (Figure 3)
<ol>
	<li>Mix 4% isoflurane with oxygen for induction of anesthesia and 2% isoflurane with oxygen for maintenance of anesthesia. Confirm adequate anesthetic depth by toe pinch.
	<ol>
		<li>Apply anti-itch ointment on eyes to protect the corneas from drying out.</li>
	</ol>
	</li>
	<li>Use encapsulated cells that are permanently labeled with artificial fluorescence protein, such as green fluorescence protein (GFP).</li>
	<li>Use a 3 ml syringe and a 20 gauge needle to inject encapsulated cells.
	<ol>
		<li>Inject 0.5 x 106 cells suspended in 0.2 ml of PBS into each iAb fat depot that is located in the intraperitoneal area between a gonad and kidney as shown in Figure 3. Use this volume of PBS and cell number for mice with an average weight of 40 g. Adjust cell number and volume in mice that have different weight or for determination of dose-dependent effects.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

Various methods have been used to encapsulate cells, including drying, extrusion, and emulsion19. In this method, the alginate beads are extruded through a needle, then coated with PLL and the alginate core will be dissolved to complete the encapsulation. Although this method has been used for years, formation of the beads with the desired size and spherical shape is still challenging. The size of the capsules is highly dependent on the viscosity of sodium alginate solution, the extruder diameter and the dista...

Increasing incidence of chronic diseases1 has stimulated studies on transplantation of therapeutic cell populations2. Syngenic or allogenic stem cells are the most commonly used cell types for these applications2. However, these treatments do not allow control of differentiation and migration of stem cells after implantation and are not cost efficient. Transplantation of genetically modified cells with beneficial functions anticipates improving the treatment of many diseases. However, genetic cell modifications are recognized by the host&#8217;s immune system, therefore, these treatments require immunosuppression3. Encapsulation of cells producing insulin has been developed by Dr. Chang4. The technique is based on encapsulation of cells in alginate droplets that are immersed into a calcium chloride solution. Alginate molecules consist of mannuronic (M) and guluronic acid (G) and can be connected by Ca2+. After gelation, the beads are suspended a poly-L-lysine (PLL) solution. During this step, PLL binds to G and M in the alginate molecules which establishes the capsule&#8217;s membrane. The porosity of the capsule&#8217;s membrane can be modulated by varying the M and PLL concentrations, the incubation time, and temperature. The binding of PLL also depends on the type and concentration of alginate. Alginate matrices crosslinked with Ca2+ ions, are unstable in the physiological environment or in common buffer solutions with high concentration of phosphate and citrate ions. These buffers can extract Ca2+ from the alginate and liquefy the core. Liquefaction of the alginate core provides space inside the capsules for cellular movement and growth. Cells encapsulated in polyanionic alginate with polycationic poly-L-lysine (APL) are impermeable for immunoglobulins but have influx of nutrients and efflux of toxins. These APL&#39;s properties enable the long term survival of encapsulated cells after transplantation into genetically different hosts. Elliott et al. reported the survival of functioning encapsulated porcine pancreatic cells in a human patient nine years after implantation5.
Encapsulation techniques can be classified into microencapsulation (3-800 &#181;m) and macroencapsulation (larger than 1,000 &#181;m). Microcapsules are more durable than macrocapsules6. Since its discovery by Dr. Chang and colleagues in 1964, microencapsulation has been widely used for the encapsulation of anabolic cells producing insulin, other hormones, and bioactive molecules7. These treatments faced several challenges in the host tissue including fibrosis and immune response8. Initially, the side effects related to the quality of biopolymers have been resolved. However, transplantation of anabolic cells still initiates side effects, such as fibrosis, as a result of hormone overproduction outside of a specialized gland.
In recent decades, obesity and type 2 diabetes has reached epidemic proportions9. More than 30% of adult people worldwide are overweight and obese10. Increased intra-abdominal (iAb) fat formation increases incidence of chronic inflammation and promotes type 2 diabetes, cardiovascular disease, certain cancers, and other morbidities11-13. Several lines of evidence suggested that pathogenesis associated with iAb fat can be averted by specific adipocytes. Recent studies have shown that transplantation of subcutaneous adipocytes into iAb region can improve metabolism and decrease obesity and insulin resistance in rodents in vivo14. Effective reduction of obesity and insulin resistance has been associated with thermogenic adipocytes capable of dissipating energy in form of heat15,16. Thermogenic modification of adipocytes can be achieved by stable transfection of genes participating in the mitochondrial proton uncoupling, such as uncoupling protein 1 (Ucp1) or of genes regulating expression of Ucp1 and other thermogenic genes15,16. Our recent studies showed that deficiency in aldehyde dehydrogenase 1 a1 (Aldh1a1) leads to the thermogenic remodeling of iAb fat that reduces obesity and insulin resistance in these mice17,18. Notably, encapsulation of thermogenic Aldh1a1 deficient (Aldh1a1-/-) preadipocytes mediates same therapeutic effect in iAb fat in obese wild type mice, suggesting new therapeutic opportunities for treatment of iAb fat18. In experimental settings, encapsulated cells enable researchers to study effects of specific cell populations in a cost effective manner19. Here we discuss the method of encapsulation of a thermogenic catabolic cell line and its laboratory and therapeutic application in a mouse model of obesity. The protocol describes three phases for microcapsule production (Figure 1): the formation of the alginate microbeads (Figure 1A), the formation of the polycationic poly-L-lysine (PLL) membranes on the surface of microbeads (Figure 1B), and the removal of the alginate cores (Figure 1C).

<table><tbody><tr><td>Encapsulation device (VAR V1)</td><td>Nisco</td><td>LIN-0042</td><td>None</td></tr><tr><td>KD scientific syringe pump</td><td>KD scientific</td><td>780100Y</td><td>None</td></tr><tr><td>Olympus microscope&amp;nbsp;</td><td>Olympus Optical</td><td>IX70-S8F2</td><td>None</td></tr><tr><td>Sodium alginate</td><td>Sigma</td><td>MKBP8122V</td><td>None</td></tr><tr><td>Poly-L-lysine hydrobromide (PLL)</td><td>Sigma</td><td>020M5006V</td><td>None</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>SLBJ2662V</td><td>None</td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma</td><td>030M0200</td><td>None</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>SLBD2595V</td><td>None</td></tr><tr><td>Mini-PROTEAN TGX Gels</td><td>Bio-Rad</td><td>456-1093</td><td>None</td></tr><tr><td>ATGL primary antibody (from rabbit)</td><td>Cell Signaling</td><td>2138S</td><td>None</td></tr><tr><td>Secondary anti body (anti rabbit)</td><td>LI-COR</td><td>926-68071</td><td>None</td></tr><tr><td>Radio-Immunoprecipitation Assay (RIPA) buffer</td><td>Boston BioProducts</td><td>D25Y6Z</td><td>None</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>RNBD2893</td><td>None</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>None</td></tr><tr><td>Cortizone 10 anti-itch ointment</td><td>Cortizone 10</td><td>C4029138</td><td>None</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-092</td><td>None</td></tr><tr><td>Newborn calf serum (CS)</td><td>Sigma</td><td>N4762</td><td>None</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma</td><td>F4135</td><td>None</td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I0516</td><td>None</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>None</td></tr><tr><td>Insulin (bovine)</td><td>Sigma</td><td>I5879</td><td>None</td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>4693159001</td><td>None</td></tr></tbody></table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Figure 1 shows that every step of microbeads production could be controlled under the microscope. Figure 2A shows how to co-culture adipocytes with a monolayer of encapsulated cells. Figure 2B is a representative example of a quantitative study using adipocyte/microcapsules co-cultures that were described in section 5. Lysates of adipocytes were analyzed using Western blot. Encapsulated cells were not analyzed in this experiment. Primary ATGL and β-actin antibodies were ...

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

The overall goal of this procedure is to demonstrate the encapsulation of a thermogenic catabolic cell line and its applications in vitro and potentially in vivo. To begin this procedure, prepare a sodium alginate cell suspension and transfer it to a five milliliter syringe. The next step is to use an encapsulation device to produce the alginate microbeads.The microbeads are then coated with a poly onic poly L lysine solution, and the alginate core is subsequently removed to form a semipermeable membrane around a cell suspension. The final step is to co-culture the encapsulated cells with other cell lines or inject them into the fat tissues of mice. Ultimately, immuno blood analysis shows higher lipolytic activity in adipocytes co cultured with aldehyde dehydrogenase, one deficient adipocyte containing microcaps.While immunohistochemistry shows successful transplantation of encapsulated cells into the fat tissues of mice, This method can provide insight into thermogenesis and apus tissue. It can also be applied to other existing animal models to provide low cost experimental model system for testing cell subset in vivo. For biomedical discoveries, All procedures should be performed in a level two biosafety cabinet with laminar flow.To begin, remove the old medium from the cell culture flask and rinse the preadipocytes with 10 milliliters of PBS. Remove the PBS and resuspend the cells in two milliliters of 0.25%tripsin EDTA. Remove a 10 microliter aliquot from the cell suspension and count cells using a hemo cytometer.According to the manufacturer's instructions, add DMEM to the remaining cells to get the ideal amount of cells and centrifuge at 480 times G at room temperature for five minutes. After determining the amount of cells needed following the directions in the protocol text, we suspend the cell pellet in an appropriate volume of a 2%sodium algin net solution. Transfer the sodium alginate cell solution to a five milliliter syringe.Remove any air bubbles in the solution and invert the syringe to create a one milliliter pocket of air. To prepare for encapsulation, place a small beaker containing 144 milliliters of a 100 millimolar calcium chloride solution. Under the needle spout of the encapsulation device, attach the electrode to the encapsulate with the tip approximately 2.5 centimeters above the surface of the calcium chloride solution.Place the syringe containing the sodium alginate cell solution Tightly in the syringe pump. Attach the rubber tube to the opening of the syringe. Push the plunger until the sodium alginate cell solution enters halfway through the tube.Adjust the voltage to 5.4 kilovolts and set the diameter to 12.06 millimeters on the syringe pump. Adjust the speed to three milliliters per hour. Start the pump, turn on the encapsulate and maintain the voltage at 5.4 kilovolts.Close the window of the hood and avoid any unnecessary vibrations until the formation of the alginic microbeads is complete. After all of the solution has passed through the needle, solidify the alginate ball shaped microbeads in the calcium chloride solution for an additional 20 minutes before coating with polyanionic polylysine or PLL. Once the sodium alginate cell ball shaped microbeads have solidified, remove the beaker containing the microbeads from the encapsulation device.Transfer the micro beads into a 50 milliliter centrifugation tube. Remove the calcium chloride solution from the microbead pellet. Wash the microbeads by adding 30 milliliters of a 0.9%sodium chloride solution and gently shaking the tube by hand.Allow the microbeads to precipitate by gravity. Then use a 25 milliliter pipette to remove the sodium chloride solution in this manner. Wash the microbeads a total of three times.Next, add the 0.05%PLL solution to the microbeads. Use 10 milliliters of PLL solution for every one milliliter of sodium alginate solution. Vortex at 1000 rotations per minute for 10 minutes, which is usually sufficient for PLL coating.After confirming the formation of the PLL coat, remove the PLL solution and wash the capsules three times with a 0.9%sodium chloride solution as shown earlier, to remove the alternate core from the PLL coated microbeads first, add 30 milliliters of a 50 millimolar sodium citrate solution to the 50 milliliter tube. Wait for five minutes or until all the sodium alginate is dissolved. Wash the capsules three times with 0.9%sodium chloride solution as shown previously.After the third wash at 20 milliliters of culture medium to the tube transfer, all the capsules containing the cells to a cell culture flask handle the encapsulated cells under standard cell culture conditions. The host cells for this procedure should be cultured in 24 well plates until confluent. Use fibroblast growth medium prepared as described in the accompanying protocol.Text for culturing preadipocytes. Transfer the microcaps into the 24 well plate containing confluent host cells add microcaps to achieve a monolayer in each well induce pre adipocyte differentiation with differentiation. Medium one incubate for 48 hours.After 48 hours, change the media to differentiation.Medium. Two, replace the media with fresh differentiation.Medium.Two. Every 48 hours proteins from the cells will be analyzed by western blot.Each step of microbead production can be observed under the microscope. An alginate microbead is shown at 20 x magnification. Prior to PLL coating after coding with PLL and after dissolving the ginnet core in a quantitative study expression of adipose triglyceride lipase or A TGL was compared among three T three L one adipocytes co cultured with acellular wild type or aldehyde dehydrogenase one A one deficient adipocyte containing microcaps.The higher expression of A TGL in adipocytes co cultured with a L DH one A one deficient to dip cyte containing microcaps indicates higher lipolytic activity. In an in vivo study, GFP labeled encapsulated cells were injected into the intraabdominal fat depo of mice. 80 days later, the injected fat pad chose discolored clusters of encapsulated cells.A paraffin embedded section of the intraabdominal fat depot stained with hemat, toin, and eosin shows clusters of implanted microcaps encapsulated cells and host adipocytes. The same section analyzed under fluorescent light shows GFP labeled transplanted cells on the in inside of round intact capsules. The expression of GFP is also an indicator of cell viability After its development.This encapsulation technique paved away for researchers in the field of metabolic diseases to explore possible treatments for obesity, insulin resistance in type two diabetes, inflammation, and cancer and modal organisms.

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Learn encapsulation of thermogenic preadipocytes for transplantation into adipose tissue depots using alginate microbeads. This method enables enhanced energy dissipation and supports obesity research applications.

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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