This work was funded by the Hollings Cancer Center&#39;s Cancer Center Support Grant P30 CA138313 pilot research funding from the National Institutes of Health (<a href="https://www.nih.gov/" target="_blank">https://www.nih.gov/</a>), and funds from the Department of Pathology &#38; Laboratory Medicine at the Medical University of South Carolina. We would like to thank Marijne Smiths for recording the interview statements.

The authors have nothing to disclose.

This protocol describes a mammary epithelial cell transplantation technique optimized for working with rats. Isolated mammary epithelial organoids from donor rats (3-5 weeks of age) are grafted into the interscapular white fat pad of recipient rats (also 3-5 weeks of age). Results can be interpreted as little as 4-6 weeks later, using light microscopy to examine the grafted tissue; however, the optimal amount of time between transplantation and sacrifice must be determined prior to implementing a full experiment. If too ...

Postnatal mammary gland development and ductal morphogenesis are processes largely influenced by hormonal signaling at the onset of puberty. In mice and rats, commonly used model organisms of mammary gland biology, this process begins around 3 weeks of age, where rapid proliferation and differentiation result in the formation of the mature parenchyma. The mature mammary gland can undergo numerous rounds of expansion and involution, a property that has been under investigation since the early 20th century. Within the context of hyperproliferation and cancer development, mammary gland transplantation techniques were developed in the 1950s1, and enhanced by the quantitative methodology contributed by Gould et al. in 19772,3,4. Refinement of the transplantation technique in rodents has contributed to major advances in understanding normal mammary gland biology that are still widely used to study the effect of various treatments and genetic manipulation on normal mammary gland development and disease states.
Many hypotheses have been generated and subsequently tested using mammary gland transplantation, first described by DeOme et al. in 19591. Experiments across several decades showed the propensity of ductal tissue excised from donor mammary glands to repopulate the entire fat pad5,6,7 and indicated that a critical component of mammary gland development resides in these epithelial structures. Later studies in mice showed that a single mammary stem cell can repopulate a cleared fat pad and contributed to the discovery of a single, common progenitor of basal and luminal mammary epithelial cells8,9,10. In line with these conclusions, it has been suggested that transplantation increases the pool of cells with multilineage-repopulating potential as a result of plasticity, allowing the grafted cells to grow a functional mammary gland7,10,11,12,13. Importantly, the use of transplantation techniques in rodents overcomes the limitations of cell culture-induced abnormalities14 and often provides results in just a matter of weeks.
While the procedure was originally described in the context of preneoplastic lesions in mice, it was soon expanded to rats and used in conjunction with the carcinogen treatment to establish multiplicity as a measure of cancer susceptibility15, but the popularity of transplantation techniques has followed the development of genetic tools for each species. Although mouse studies incorporating transplantation have contributed many translational findings, the parenchyma of the rat mammary gland resembles the human more closely16,17 and offers distinct advantages for studying estrogen receptor-positive (ER+) breast cancer. Mammary tumors are inducible in both species, but they differ in terms of hormone sensitivity and gene expression profiles. A primary difference is that rat mammary tumors express and depend on the function of ovarian and pituitary hormone receptors, namely, estrogen and progesterone (PR), similar to the luminal-A subtype of human breast cancer. Indeed, mammary epithelial cell transplantation, as described in this protocol, has been used to study genetic variants involved in breast cancer and determine the cellular autonomy of effects on mammary epithelial cells18.
In addition to the tumor biology, the ductal epithelium of the normal rat mammary gland exhibits a higher level of branching and is flanked by a thicker layer of stroma than the mouse. The importance of the stroma is well-documented in mammary epithelial transplantation studies. Mammary epithelium must interact with fatty stroma, and ideally its own mesenchyme, to undergo its characteristic morphogenesis19,20. Grafting tissue into a recipient mammary gland provides an optimal environment; however, the presence of endogenous epithelium can interfere with results. Preclearing the mammary gland of endogenous epithelium is commonly performed in mouse transplantation assays and requires surgical excision of endogenous mammary tissue and/or removal of the nipple1,21,22. Although possible, preclearing the mammary epithelium in post-weanling rats is not as widely-performed, mainly due to the ineffectiveness of clearing the growing mammary tree in post-weanling rats. Since it has been shown that regions of adipose tissue elsewhere in the body could support the growth of transplanted mammary epithelium21,23,24, the process of preclearing can be easily avoided in rats by grafting tissue into the interscapular white fat pad.
The transplantation method described in this paper involves the injection of enzymatically dissociated mammary gland organoids (fragments of mammary ductal epithelium and other cells types capable of morphogenesis) or monodispersed cells into the interscapular fat pad in inbred, isogenic or congenic strains of laboratory rats2. Because the interscapular fat pad is normally devoid of mammary tissue, it provides a suitable environment for multiple transplantation sites without the need to pre-clear endogenous epithelium. As a result, the host animal&#39;s endogenous, abdominal-inguinal mammary glands are not subject to surgical manipulation, develop normally, and cannot interfere with interpretation of results. Additionally, the intact mammary glands can be used for comparison to evaluate host versus donor effects on the mammary epithelium development and tumorigenesis18,25. Although repopulation of the mammary gland from a single stem cell is available for mice, it has not yet been developed for rat, mainly due to the lack of availability of antibodies to select for rat mammary stem cells25,26,27. Despite this, transplantation of monodispersed mammary epithelial cells to quantify repopulating potential can be successfully performed, and those cells will develop normally when grafted into the appropriate framework2,3,4. While organoids are good for many purposes, monodispersed cells are required for quantitative applications, for example, to determine the number of mammary epithelial cells required for the cancer initiation following ionizing radiation treatment28 or for comparing characteristics of flow cytometrically selected mammary epithelial cell populations29.
To date, the procedure described here is the most robust method of performing mammary gland transplantation in the rat with an overall goal of studying mammary gland development and mechanisms underlying breast cancer development. Often, the donor and/or recipient animals are exposed to different variables before, during, or after the epithelial transplantation. Examples include single gene studies involving chemical carcinogenesis30, radiation28,31,32, genetic manipulation of host/donor genome18, and hormonal manipulation12. A major advantage of the enzymatic dissociation described in this protocol is the opportunity to isolate epithelial organoids or monodispersed cells for complementary experiments involving flow cytometry, 3-D culture, gene editing, and more. Future applications of this technique will include additional manipulation of donor and/or host tissue with genetic engineering. For example, donor cells can be genetically altered ex vivo at any chosen genomic locus using the CRISPR-Cas9 gene editing system. Similarly, recipient rats can also be genetically altered to study the interaction between donor and recipient engineered genetic factors.

<table><tbody><tr><td>0.2 &amp;micro;M syringe filters</td><td>Fisher Scientific</td><td>09-715G</td><td>sterile-filtering collagenase digestion media</td></tr><tr><td>1.5 - 2.0 mL microcentrifuge tubes (sterile)</td><td>Fisher Scientific</td><td>05-408-129</td><td>containing resuspended cells and/or brain homogenate mixture</td></tr><tr><td>100 &amp;micro;M cell strainers</td><td>Corning</td><td>431752</td><td>filtering brain homogenate</td></tr><tr><td>100 uL gastight syringes with 25 gauge needles</td><td>Hamilton</td><td>81001 &amp;amp; 90525</td><td>For injecting graft mixture into recipient animals (1 per donor genotype/condition)</td></tr><tr><td>1000 uL pipette tips + pipette</td><td>-</td><td>-</td><td>transferring cells/mixtures/tissue</td></tr><tr><td>15 mL polypropylene tube</td><td>Falcon (Corning)</td><td>352196</td><td>brain homogenate mixture storage, or cell : homogenate mixture for transplantation</td></tr><tr><td>40 &amp;micro;M cell strainers</td><td>Corning</td><td>431750</td><td>filtering organoids after washing the cell pellet</td></tr><tr><td>50 mL polypropylene tubes</td><td>Fisher Scientific</td><td>05-539-6</td><td>for collagenase digestion of donor mammary gland tissue</td></tr><tr><td>60 mm dishes</td><td>Thermo Scientific</td><td>130181</td><td>for mincing tissue</td></tr><tr><td>Alum Potassium Sulfate</td><td>Sigma-Aldrich</td><td>243361/237086</td><td>staining mammary gland whole mount slides</td></tr><tr><td>Anesthesia vaporizer for veterinary use</td><td>-</td><td>-</td><td>follow institutional protocol</td></tr><tr><td>Beta-dine or iodine</td><td>-</td><td>-</td><td></td></tr><tr><td>Borosilicate glass culture tube for homogenization</td><td>Fisher Scientific</td><td>14-961-26</td><td>for homogenization of brain (use appropriate tube for homogenizer)</td></tr><tr><td>Carmine</td><td>Sigma-Aldrich</td><td>C6152/1022</td><td>staining mammary gland whole mount slides</td></tr><tr><td>Cell counting apparatus</td><td>-</td><td>-</td><td></td></tr><tr><td>Clean animal cages for recovery</td><td>-</td><td>-</td><td>follow institutional protocol</td></tr><tr><td>Collagenase Type 3</td><td>Worthington Biochemical Corp.</td><td>LS004183</td><td>enzymatic digestion of minced mammary gland tissue from donor rats</td></tr><tr><td>deionized water</td><td>-</td><td>-</td><td>for chemical solutions</td></tr><tr><td>DMEM/F12</td><td>GIBCO</td><td>11320033</td><td>for mincing tissue, collagenase digestion media and resuspending epithelial cell mixtures</td></tr><tr><td>EDTA</td><td>-</td><td>-</td><td>monodispersion mixture</td></tr><tr><td>Ethanol, 200 Proof</td><td>Decon Labs</td><td>2705/2701</td><td>mammary whole mount slide fixative, mammary whole mount slide washes, cleaning surgical incision sites (diluted)</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>-</td><td>inactivation solution</td></tr><tr><td>Gauze</td><td>-</td><td>-</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Fisher Scientific</td><td>A38-212</td><td>use for mammary whole mount slide fixative (1:4 glacial acetic acid in 100% ethanol)</td></tr><tr><td>HBSS</td><td>GIBCO</td><td>-</td><td>monodispersion mixture</td></tr><tr><td>Heating pads</td><td>-</td><td>-</td><td>follow institutional protocol</td></tr><tr><td>Ice buckets (x2)</td><td>-</td><td>-</td><td></td></tr><tr><td>Incubator with orbital rotation</td><td>-</td><td>-</td><td>must be capable of maintaining 37&amp;deg;C, shaking at 220-225 RPM (for collagenase digestion of mammary tissue)</td></tr><tr><td>Isoflurane anesthesia</td><td>-</td><td>-</td><td>follow institutional protocol</td></tr><tr><td>Light microscope or digital camera</td><td>-</td><td>-</td><td>visualizing whole mounted mammary epithelium and/or acquiring images</td></tr><tr><td>Mechanical homogenizer</td><td>Fisher Scientific</td><td>-</td><td>TissueMiser or alternative models</td></tr><tr><td>Mineral oil, pure</td><td>Sigma-Aldrich/ ACROS Organics</td><td>8042-47-5</td><td>long-term storage of cleared mammary gland whole mounts</td></tr><tr><td>Oxygen tanks for anesthesia vaporizer</td><td>-</td><td>-</td><td>follow institutional protocol</td></tr><tr><td>Paper towels or delicate task wipes</td><td>-</td><td>-</td><td></td></tr><tr><td>Positively-charged microscope slides</td><td>Thermo Scientific</td><td>P4981-001</td><td>mammary gland tissue whole mounts</td></tr><tr><td>Postoperative analgesic</td><td>-</td><td>-</td><td>Institutional protocol</td></tr><tr><td>Scale</td><td></td><td></td><td>body weight measurements of animals, proper dosing of pain medication</td></tr><tr><td>Shaver</td><td>-</td><td>-</td><td>electric clippers, or other</td></tr><tr><td>Staining jars</td><td>-</td><td>-</td><td>minimum of 1 per chemical wash, size appropriate for the number of slides, glass preferred</td></tr><tr><td>Sterile field drapes</td><td>IMCO</td><td>4410-IMC</td><td>used during transplantation</td></tr><tr><td>Sterile scissors and forceps x3 (autoclaved)</td><td>-</td><td>-</td><td>autoclave surgical tools used for donors and recipients</td></tr><tr><td>Syringes: 5 mL (or greater)</td><td>-</td><td>-</td><td>for sterile filtration of collagenase digestion media</td></tr><tr><td>Trypsin</td><td>Worthington</td><td></td><td>monodispersion mixture</td></tr><tr><td>Waste collection receptacle for liquids (poured or aspirated)</td><td>-</td><td>-</td><td></td></tr><tr><td>Wound clip applier, clips, and removal tool</td><td>Fine Science Tools</td><td>12020-00</td><td>Closing the skin incision over the interscapular white pad pad</td></tr><tr><td>Xylenes</td><td>Fisher Scientific</td><td>X3S-4</td><td>clearing mammary gland whole mount slides after staining</td></tr></tbody></table>

rat mammary epithelial cell transplantation into the interscapular white fat pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

Donor and recipient mammary glands 
The steps to isolate and prepare rat mammary epithelial cells for transplantation are shown in Figure 1A. At 4 weeks of age, the endogenous mammary gland of the donor rat has begun maturation and epithelium can be visualized on whole mounted slides stained with alum carmine (Figure 1B). One donor rat at this ag...

Watch this Scientific Journal Video about Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad at JoVE.com

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

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Research

JoVE Journal

Cancer Research

7.7K Views.  Medical University of South Carolina. This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

Video: Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Medicine

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Developmental Biology

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Biology

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioengineering

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...