The authors have no funding to declare.

NOTE: The University of Stirling NHS, Invasive, or Clinical Research Committee approved the biopsy procedure described below. All research studies using this procedure must be approved by the appropriate independent ethics committee. The biopsy taker must have completed formal training in the described technique in accordance with their institution&#39;s requirements. Typically, this involves observing a demonstration of the described adipose tissue biopsy technique by a board-certified physician, followed by supervised practice. Once the trainee has performed 10 practice adipose tissue biopsies on volunteer subjects under supervision, they will be examined by a board-certified physician to ensure good knowledge and practice of the procedure. The board-certified physician then provides the individual with a signed examination form.
1. Laboratory room preparation
<ol>
	<li>Ensure that the laboratory has an appropriately private room with clean, wipeable non-porous surfaces and a clean, comfortable (preferably non-porous) bed on which the participant may lie supine. Clean all required surfaces for the biopsy procedure using 70% ethanol spray and clean paper towels. Provide clean pillows or cushions to support the participant if required.</li>
	<li>Keep appropriate sharps disposal bins and biohazard waste bags within easy reach of the area where the biopsy is being performed and within easy reach of the person taking the biopsy.</li>
	<li>Prepare the equipment required for the procedure and set up on a freshly cleaned general medical trolley prior to the participant arriving to the laboratory (Figure 1). For a complete list of consumables required, see the Table of Materials.</li>
</ol>
2. Participant preparation
<ol>
	<li>Ensure that all participants provide written informed consent prior to undergoing the procedure in accordance with protocols required by the institution&#39;s independent ethics committee. Additionally, ask the participants to complete a written questionnaire to ensure they are not allergic to any materials used in the procedure (namely, nickel, chromium, local anesthetic, iodine, shellfish, and plasters).</li>
	<li>Confirm the identity of the participant. Ensure the participant understands the procedure to be carried out and potential secondary effects, including bruising, pain, and infection (Table 1). Gather verbal consent in addition to previously obtained written informed consent.</li>
	<li>Describe to the participant how the procedure will be carried out, with emphasis on how the administration of the anesthetic and biopsy itself will feel. Ensure that the participant is comfortable with proceeding. 
	NOTE: Local subcutaneous anesthetic will produce a stinging sensation, similar to a bee sting of short duration. Many participants report the anesthetic administration as the most uncomfortable part of the technique. Once the anesthetic has taken effect, the participant should feel no more than a slight tugging sensation during the biopsy.</li>
	<li>Ensure that the participant has no allergies to the local anesthetic (specifically from the amino-amide type, if using lidocaine or similar), certain metals (nickel and chromium), and shellfish (if using iodine-based solutions). Additionally, ensure that the participants are not taking any form of anticoagulant medication.</li>
	<li>Provide the participant with an opportunity to go and empty their bladder if required, to ensure they do not have to interrupt the procedure or experience undue discomfort in step 4.1.</li>
</ol>
3. Biopsy procedure - instructions for the biopsy taker
<ol>
	<li>Once the participant is lying in a supine position, identify the biopsy site approximately 5-10 cm lateral to the umbilicus. 
	NOTE: If the participant is to undergo multiple biopsies on the same day, identify biopsy sites on opposing sides of the umbilicus for each biopsy. This will ensure maximal distance between each biopsy site.</li>
	<li>Wash hands with soap and warm water according to standard medical guidelines4.</li>
	<li>Place the sterile sheet on the cleaned trolley or work area, taking care to only touch the outer edges of the sheet.</li>
	<li>Put on sterile surgical gloves using proper aseptic technique. Have the assistant open the rest of the equipment in such a way that it drops onto the prepared sterile sheet without touching/contaminating the equipment. Ensure that the assistant takes care not to touch items when removing tools from their sterile wrappings.</li>
	<li>Instruct the assistant to dispense a small amount of iodine-based solution on some sterile gauze (without oversaturating the gauze) on the work surface.</li>
	<li>Sterilize approximately 5-10 cm2 around the chosen biopsy site using the sterile gauze and iodine-based solution. Ensure the skin is cleaned in a spiraling motion moving outward from the proposed biopsy site. Repeat the skin cleaning procedure twice. Remove excess liquid (e.g., running off sterile area) by wiping with fresh sterile gauze.</li>
	<li>Along with the assistant, verbally confirm the content of the local anesthetic vial (2% lidocaine in this protocol) and that this is within its expiry date. Instruct the assistant to hold the opened vial upside down and draw 5 mL of local anesthetic into a syringe, using a 21 G needle. Dispose of the needle into the sharps bin, and ensure the syringe is free of air bubbles.</li>
	<li>Apply a 26 G needle to the syringe and expel any air bubbles. Gently pinch the abdominal skin and adipose tissue, moving it away from the abdominal wall. Then, insert the needle horizontally into the subcutaneous tissue at an angle no greater than 10&#176; relative to the surface of the skin.
	<ol>
		<li>Withdraw the syringe&#39;s plunger an additional 0.5 mL (to ensure the needle is not in a blood vessel). If blood appears in the syringe, withdraw and reinsert the needle at a different angle.</li>
		<li>Raise a bleb of 2-4 mm diameter to anesthetize the insertion area.</li>
		<li>Advance the needle into the subcutaneous tissue and administer ~1 mL of lidocaine in a fan-shaped pattern (Figure 2), taking care to withdraw the plunger each time before injecting the anesthetic.</li>
		<li>Remove and dispose of the 26 G needle, apply a 21 G needle to the syringe, expel any air bubbles, and administer the remaining ~4 mL of lidocaine in a fan-shaped pattern (Figure 2), taking care to withdraw the plunger each time before injecting the anesthetic.</li>
	</ol>
	</li>
	<li>Wait approximately 5 min for the local anesthetic to take effect. Use a sterile scalpel to gently prod the biopsy area to i) ensure the local anesthetic has taken effect and ii) identify the boundaries of the anesthetized area. If necessary, wait an additional minute or two and reassess.</li>
	<li>Once satisfied that the local anesthetic is working, gently pinch the skin and adipose tissue (as in step 3.8) and use a sterile scalpel to make a small 1-2 mm puncture in the skin. 
	NOTE: This only needs to be large enough to ease the entry of the 14 G needle and must be small enough that no suture is required to close it. It is common for some bleeding to occur from this point onwards, which can be controlled with a piece of sterile gauze.</li>
	<li>First, apply a 14 G needle to a 5 or 10 mL syringe. Then, while gently pinching the skin and adipose tissue, gradually insert the needle through the puncture into the adipose tissue approximately centrally in the anesthetized area and at an angle no greater than 10&#176; relative to the surface of the skin. 
	NOTE: For all cases of needle advancement in step 3.11, a syringe angle of no greater than 10&#176; must be maintained.
	<ol>
		<li>Apply suction by withdrawing the plunger to approximately the 2.5 mL mark. Take the biopsy by moving the needle in a quick backwards and forwards motion to slice fragments of adipose tissue. After approximately 30 s, twist the needle and syringe through 90&#176; and repeat this procedure to break up the fragments of adipose tissue, which are then aspirated into the syringe by the suction. 
		&#8203;NOTE: Other syringe sizes can be used. It is essential that the researcher selects a syringe size that permits both a good grip on the syringe and to comfortably maintain plunger retraction for maintenance of the vacuum. Locking syringes are available that maintain the vacuum, which can improve needle control and reduce perceived difficulty for the biopsy taker 5.</li>
		<li>After approximately 45-60 s of step 3.11.1, remove the needle and empty the syringe content onto a layer of gauze covering a weighing boat. Ensure that the lumen of the needle is facing down to avoid potential blood spatter.</li>
		<li>Repeat steps 3.11.1 and 3.11.2 for a maximum of 3 times. Check that the participant is content to proceed before each repeat of the above procedure.</li>
		<li>Whilst performing steps 3.11.1 and 3.11.2, instruct the assistant to process and prepare the samples for analysis/storage (see section 5).</li>
	</ol>
	</li>
</ol>
4. Post-biopsy procedure
<ol>
	<li>Once a satisfactory sample (i.e., ~200 mg) of adipose tissue has been obtained, place 1-2 layers of sterile gauze over the puncture wound, then place an ice pack over these, and apply firm pressure for approximately 10 min to induce hemostasis.</li>
	<li>When hemostasis has occurred, wipe away any iodine-based solution/dried blood with sterile gauze, and apply an adhesive wound dressing with absorbent pad to the site. Check that the participant feels well and provide verbal and written instructions on biopsy site aftercare.
	<ol>
		<li>Emphasize that the participants will likely exhibit some bruising for the next few days. Inform them that this may be substantial, although it is minimized by the ice pack in step 4.1 and will resolve without lasting effects.</li>
		<li>Recommend that should the participants feel any discomfort/pain once the anesthetic has worn off, they should take analgesics such as paracetamol following the instructions on the packet but refrain from taking analgesics that have anticoagulant activities (e.g., ibuprofen or aspirin).</li>
		<li>Explain that swelling, redness, or discharge from the biopsy site are indications of infection. In the unlikely event that these signs or symptoms occur, instruct the participant to urgently seek medical advice from a doctor or local Accident &#38; Emergency unit. Inform the participant that if they seek medical advice, they must also notify the research team. 
		NOTE: As research staff, neither the biopsy taker nor the assistant can provide medical advice or treatment; however, it is important that the research team are aware of and record all instances of complications resulting from the biopsy procedure.</li>
		<li>Recommend that participants should avoid swimming or overly strenuous activity for 48 h until the site of incision has closed.</li>
	</ol>
	</li>
	<li>Clear away any used sharps and contaminated materials into designated sharps and/or clinical waste containers.</li>
	<li>Clean all surfaces used in the biopsy procedure using 70% ethanol spray and clean paper towels. Place disposable and non-disposable items of bedding in appropriate clinical bags for disposal or cleaning, respectively.</li>
</ol>
5. Sample processing - instructions for the assistant
<ol>
	<li>Use sterile tweezers and 0.9% saline to rinse the adipose tissue sample to remove visible contaminants (i.e., blood, vasculature). Then, weigh the adipose tissue samples using digital scales. Split the tissue into appropriately sized pieces for downstream analysis and place them into appropriate storing tubes using sterile tweezers. Immerse the tubes containing the adipose tissue biopsies in liquid nitrogen at -190 &#176;C to flash-freeze until the samples are stored at -80 &#176;C. 
	NOTE: The assistant must complete sample processing as quickly as possible, typically within 3 min of sample aspiration, to minimize potential sample degradation.</li>
</ol>

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The authors have no conflicts of interest to declare.

The described protocol and associated video provide a step-by-step overview of a mini-liposuction technique to obtain subcutaneous adipose tissue samples from the abdominal area. This research group has performed a total of 124 biopsies over the course of 19 months with no adverse effects in participants. The procedure is safe and associated with minimum risk to participants or the biopsy team, provided that the described safety measures are followed. Aseptic technique (including opening and dispensing of sterile equipme...

Adipose tissue can provide useful information on the metabolic function of humans. Human subcutaneous adipose tissue is readily accessible. A technique for subcutaneous adipose tissue extraction was first described in the mid-80s1; since then, the initial protocol has been improved to increase the yield and improve study participant tolerability. Subcutaneous adipose tissue can be obtained from numerous sites, most commonly from the glutei1 and abdominal area2. Samples from the latter may be more desirable as they provide more valuable information in metabolic disease-related contexts3.
Subcutaneous adipose tissue biopsy using the mini-liposuction method can be safely and efficiently performed in a non-clinical setting. Following appropriate training by a board-certified physician and using strict aseptic technique, researchers can routinely perform these biopsies with minimal risk to both participant and investigators. The biopsy team must consist of at least 2 individuals: the person who will perform the biopsy and an assistant.
The person responsible for the biopsy is tasked with confirming the participant&#39;s identity, checking the participant can safely undergo the procedure (see protocol steps 2.1-2.4 below), ensuring the participant is comfortable throughout the procedure, ensuring sterile technique is maintained throughout the procedure, carrying out the procedure, and providing the participant with verbal and written after-care procedures. The assistant&#39;s role is to handle and rapidly process the adipose tissue obtained for later analysis and/or storage. The assistant also helps by being the &#34;non-sterile hands&#34; and ensuring the participant is at ease throughout the procedure. The purpose of this video and paper is to describe the step-by-step biopsy procedure to safely obtain subcutaneous adipose tissue from the abdominal area.

<table><tbody><tr><td>Item: 14 G needle&lt;br/&gt; 14 G x 3 1/8&quot; 210 mm x 80 mm</td><td>B Braun</td><td>4665473</td><td>Per biopsy: 1</td></tr><tr><td>Item: 21 G needle&lt;br/&gt; 21 G x 1 1/2&quot; 0.8 mm x 38 mm</td><td>Terumo</td><td>AN*2138R1</td><td>Per biopsy: 2</td></tr><tr><td>Item: 26 G&amp;nbsp; needle&lt;br/&gt; 26 G x 1/2&quot; microlance needle 0.45 mm x 13 mm</td><td>BD</td><td>303800</td><td>Per biopsy: 2</td></tr><tr><td>Item: 5 mL syringe&lt;br/&gt; 5 mL luer</td><td>DB plastipak</td><td>302187</td><td>Per biopsy: 2</td></tr><tr><td>Item: Adhesive wound dressing&lt;br/&gt; Opsite Post-Op Dressing 9.5 x 8.5</td><td>Smith &amp;amp; Nephew</td><td>6600709</td><td>Per biopsy: 1</td></tr><tr><td>Item: Disposable sterile scalpel&lt;br/&gt; Disposable Scalpel Sterile Blade no. 10</td><td>Swann Morton</td><td>/0501</td><td>Per biopsy: 1</td></tr><tr><td>Item: Icepack&lt;br/&gt; BlueDot Reusable Hot/Cold Pack 26.5 cm x 13.0 cm</td><td>NuCare</td><td>F711</td><td>Per biopsy: 1</td></tr><tr><td>Item: Iodine based antiseptic&lt;br/&gt; Videne antiseptic solution</td><td>Ecolab Videne</td><td>3030440</td><td>Per biopsy: q.s</td></tr><tr><td>Item: Lidocaine 2% w/o epinephrine&lt;br/&gt; Lidocaine 2% injection 5 mL</td><td>B Braun</td><td>3558553</td><td>Per biopsy: 5 mL</td></tr><tr><td>Item: Non-sterile gloves&lt;br/&gt; Starguard sensitive powder free nitrile gloves</td><td>Starguard</td><td>SG-N-S</td><td>Per biopsy: pair</td></tr><tr><td>Item: Sodium chloride 0.9%&lt;br/&gt; Sodium chlride 0.9% w/v intravenous infusion BP</td><td>BBraun</td><td>S8004-5384</td><td>Per biopsy: q.s.</td></tr><tr><td>Item: Stabilization solution*&lt;br/&gt; RNAlater Stabilization Solution</td><td>ThermoFisher Scientific</td><td>AM7020</td><td>Per biopsy: q.s</td></tr><tr><td>Item: Sterile forceps&lt;br/&gt; Sterile forceps</td><td>Rocialle</td><td>RML109-006</td><td>Per biopsy: 1</td></tr><tr><td>Item: Sterile gauze swabs&lt;br/&gt; Non woven swabs sterile 7.5 x 7.5 cm</td><td>Prestige</td><td>1860</td><td>Per biopsy: 5</td></tr><tr><td>Item: Sterile gloves&lt;br/&gt; Prestige soft vinyl sterile powder free medical gloves</td><td>Prestige</td><td>S: P4301&lt;br/&gt; M:P3302&lt;br/&gt; L:P3301</td><td>Per biopsy: pair</td></tr><tr><td>Item: Sterile Microcentrifuge tubes&lt;br/&gt; 1.5 mL Sterile Microcentrifuge Tubes</td><td>StarLab</td><td>I1415-5510</td><td>Per biopsy: q.s</td></tr><tr><td>Item: Sterile sheet&lt;br/&gt; Paper plain white 90 x 90 cm</td><td>Rocialle</td><td>RML 126-216</td><td>Per biopsy: 1</td></tr><tr><td>Item: Weighing boat&lt;br/&gt; Diamond shape weigh boats</td><td>Heathrow Scientific</td><td>HS1427C</td><td>Per biopsy: 1</td></tr><tr><td>* denotes optional materials</td><td></td><td></td><td></td></tr></tbody></table>

human subcutaneous adipose tissue sampling using a mini-liposuction technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The described adipose tissue biopsy procedure is an efficient and low-risk technique for researchers to obtain subcutaneous adipose tissue samples from human volunteers. We performed 39 subcutaneous adipose tissue biopsies using the described procedure in 11 healthy, normal weight females (age, 27.4 &#177; 3.3 years; body mass index (BMI), 22.6 &#177; 1.5 kg.m2). All participants attended the laboratory between 07:00 and 10:00 following an 8-12 h fasting period. Sample yield using this adipose tissue biopsy pr...

Watch this Scientific Journal Video about Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique at JoVE.com

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...

human-subcutaneous-adipose-tissue-sampling-using-mini-liposuction

Research

JoVE Journal

Medicine

2.9K Views.  University of Stirling. Deze techniek is bijzonder geschikt voor elke studie die een voor- en nastudieontwerp implementeert of een specifieke populatie wil karakteriseren en geen overmatige hoeveelheden lichaamspostweefsel vereist. De belangrijkste voordelen zijn dat de techniek relatief kosteneffectief en bijna pijnvrij is. U kunt meerdere biopten uitvoeren op dezelfde deelnemer op dezelfde dag.Deze techniek kan helpen bij het begrijpen van de fysiologie van vetweefsel, in het bijzonder het begrijpen van zie...

Video: Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Developmental Biology

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Biochemistry

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

Behavior

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 ...

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 ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Bioengineering

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

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

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

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.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Biology

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium (2H)-Labeling Approach

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat mass or distribution are regulated by changes in the synthesis and breakdown (i.e., turnover) of adipose cells and triacylglycerols. Evidence suggests that the manner and magnitude of subcutaneous adipose tissue expansion (i.e., hypertrophy vs. hyperplasia) and turnover can influence metabolic health, as adipogenesis has been implicated in the pathogenesis of obesity and related diseases. Despite the potential role of adipose turnover in human health, there is a lack of knowledge about the in vivo kinetics of adipose cells. This is due, in part, to the slow turnover rate of the cells in adipose tissue and the practical complexity of directly labeling their metabolic precursors in vivo. Herein, we describe methods to measure in vivo adipose kinetics and turnover rates in humans through the consumption of deuterium (2H)-labeled water. The incorporation of 2H into the deoxyribonucleotide moieties of DNA in pre-adipocytes and adipocytes provides an accurate measure of cell formation and death (adipose turnover). Overall, this is an innovative approach to measuring in vivo adipose kinetics and represents a substantive departure from other in vitro assessments.

Measuring In Vivo Adipose Tissue Kinetics in Humans Using the Deuterium 2H-Labeling Approach

Presented here is a protocol to measure in vivo adipose tissue kinetics in humans using the deuterium (2H)-labeling method.

White adipose tissue is a highly plastic organ that is necessary to maintain whole-body energy homeostasis. The adipose tissue mass and changes in the fat ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

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