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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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&#39;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

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 &#946;-actin antibodies ...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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 ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Research

JoVE Journal

Medicine

9.2K Views.  The Ohio State University. 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.

Video: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...