Cell Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 587 Views Feb 20, 2024

Inflammatory bowel disease (IBD) is characterized by an aberrant immune response against microbiota. It is well established that T cells play a critical role in mediating the pathology. Assessing the contribution of each subset of T cells in mediating the pathology is crucial in order to design better therapeutic strategies. This protocol presents a method to identify the specific effector T-cell population responsible for intestinal immunopathologies in bone marrow–engrafted mouse models. Here, we used anti-CD4 and anti-CD8β depleting antibodies in bone marrow–engrafted mouse models to identify the effector T-cell population responsible for intestinal damage in a genetic mouse model of chronic intestinal inflammation..

Key features

• This protocol allows addressing the role of CD4+ or CD8αβ+ in an engrafted model of inflammatory bowel disease (IBD).

• This protocol can easily be adapted to address the role of other immune cells or molecules that may play a role in IBD.

0 Q&A 744 Views Jan 20, 2024

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions. The core elements of our method are the sequential transplantation of fibroblasts followed by keratinocytes seeded into a fibrin-based hydrogel in a silicone chamber. We optimized the fibrin gel formulation, timing for gel polymerization in vivo, cell culture conditions, and seeding density to make a robust and efficient grafting protocol. Using this approach, we can successfully engraft as few as 1.0 × 106 fresh and 2.0 × 106 frozen-then-thawed keratinocytes per 1.4 cm2 of the wound area. Additionally, it was concluded that a successful layer-by-layer engraftment of skin cells in vivo could be obtained without labor-intensive and costly methodologies such as bioprinting or engineering complex skin equivalents.

Key features

• Expands upon the conventional skin chamber assay method (Wang et al., 2000) to generate high-quality skin grafts using a minimal number of cultured skin cells.

• The proposed approach allows the use of frozen-then-thawed keratinocytes and fibroblasts in surgical procedures.

• This system holds promise for evaluating the functionality of skin cells derived from induced pluripotent stem cells and replicating various skin phenotypes.

• The entire process, from thawing skin cells to establishing the graft, requires 54 days.

Graphical overview

Generation of a human skin equivalent on an immunodeficient mouse using a fibrin-based grafting system. A schematic of the protocol is shown. Cultured keratinocytes and fibroblasts resuspended in a fibrin-based gel are delivered as layers into a silicon chamber inserted underneath the skin of an immunocompromised mouse. First, a fibrin gel containing encapsulated fibroblasts (up to 2 × 106 per 1.4 cm2 wound) is delivered into the chamber and allowed to solidify for 15 minutes. Second, a fibrin gel containing 1.0–2.0 × 106 keratinocytes is applied on top of the fibroblast layer. On day 7 post-grafting, the chamber is removed, and the wound with the graft is allowed to heal for 4–5 weeks. During healing, a scab forms and eventually falls off. By day 54, the graft is fully established.

0 Q&A 477 Views Jan 20, 2024

Cell-based liver therapies utilizing functionally stabilized engineered hepatic tissue hold promise in improving host liver functions and are emerging as a potential alternative to whole-organ transplantation. Owing to the ability to accommodate a large ex vivo engineered hepatocyte mass and dense vascularization, the mesenteric parametrial fat pad in female nude mice forms an ideal anatomic microenvironment for ectopic hepatocyte transplantation. However, the lack of any reported protocol detailing the presurgical preparation and construction of the engineered hepatic hydrogel, fat pad surgery, and postsurgical care and bioluminescence imaging to confirm in vivo hepatocyte implantation makes it challenging to reliably perform and test engraftment and integration with the host. In this report, we provide a step-by-step protocol for in vivo hepatocyte implantation, including preparation of hepatic tissue for implantation, the surgery process, and bioluminescence imaging to assess survival of functional hepatocytes. This will be a valuable protocol for researchers in the fields of tissue engineering, transplantation, and regenerative medicine.

Key features

• Primary human hepatocytes transduced ex vivo with a lentiviral vector carrying firefly luciferase are surgically implanted onto the fat pad.

• Bioluminescence helps monitor survival of transplanted hepatic tissue over time.

• Applicable for assessment of graft survival, graft-host integration, and liver regeneration.

Graphical overview

0 Q&A 296 Views Mar 20, 2023

Adult stem cells not only maintain tissue homeostasis but are also critical for tissue regeneration during injury. Skeletal stem cells are multipotent stem cells that can even generate bones and cartilage upon transplantation to an ectopic site. This tissue generation process requires essential stem cell characteristics including self-renewal, engraftment, proliferation, and differentiation in the microenvironment. Our research team has successfully characterized and isolated skeletal stem cells (SSCs) from the cranial suture called suture stem cells (SuSCs), which are responsible for craniofacial bone development, homeostasis, and injury-induced repair. To assess their stemness features, we have demonstrated the use of kidney capsule transplantation for an in vivo clonal expansion study. The results show bone formation at a single-cell level, thus permitting a faithful assessment of stem cell numbers at the ectopic site. The sensitivity in assessing stem cell presence permits using kidney capsule transplantation to determine stem cell frequency by limiting dilution assay. Here, we described detailed protocols for kidney capsule transplantation and limiting dilution assay. These methods are extremely valuable both for the evaluation of skeletogenic ability and the determination of stem cell frequency.

1 Q&A 1307 Views Feb 5, 2023

While wound healing in humans occurs primarily through re-epithelization, in rodents it also occurs through contraction of the panniculus carnosus, an underlying muscle layer that humans do not possess. Murine experimental models are by far the most convenient and inexpensive research model to study wound healing, as they offer great variability in genetic alterations and disease models. To overcome the obstacle of contraction biasing wound healing kinetics, our group invented the splinted excisional wound model. While other rodent wound healing models have been used in the past, the splinted excisional wound model has persisted as the most used model in the field of wound healing. Here, we present a detailed protocol of updated and refined techniques necessary to utilize this model, generate results with high validity, and accurately analyze the collected data. This model is simple to conduct and provides an easy, standardizable, and replicable model of human-like wound healing.

0 Q&A 1043 Views Dec 5, 2022

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies.

Graphical abstract

0 Q&A 1862 Views Jun 5, 2022

Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo. The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities.

Graphical abstract:

Schematic overview for assessment of stem cells in spleen by transplantation. Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.

0 Q&A 4665 Views Sep 5, 2020
Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish neuronal connectivity with the host circuitry.
0 Q&A 3040 Views Jul 5, 2020
For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several In vitro assays were developed, such as mammospheres formation assay, detection of stem cell markers by mRNA expression or flow cytometry and so on. However, the capacity of reconstruction of whole mount in the cleared fat pad of recipient female mice is a golden standard to estimate the stemness of the cells. Here we described a step-by-step protocol for in vivo mammary gland formation assay, including preparation of “cleared” recipients and mammary cells for implantation, the surgery process and how to assess the experimental results. Combined with manipulation of mammary cells via gene editing and /or drug treatment, this protocol could be very useful in the researches of mammary stem cells and mammary development.
0 Q&A 6484 Views Feb 5, 2019
Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes.

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