Cell Biology

Obtaining articular cartilage-derived cells (chondroprogenitors) by explant methodology is a reliable approach for isolating migratory progenitor cells that retain strong chondrogenic potential. This method allows cells to emerge naturally from small cartilage fragments without enzymatic digestion. The procedure consists of plating cartilage explants on a plastic surface with culture medium, from which cells subsequently migrate and adhere to the substrate. Compared with enzymatic isolation, the explant approach minimizes cellular stress and better reproduces the physiological microenvironment of cartilage tissue. This protocol can be applied to both osteoarthritic and non-osteoarthritic samples, enabling comparative studies on disease-related phenotypic differences. Overall, this technique offers a reproducible, straightforward, and minimally invasive strategy for obtaining functional chondroprogenitor cells suitable for cartilage regeneration research.

Breast cancer remains one of the most prevalent and deadly malignancies affecting women worldwide. Its progression and metastatic behavior are driven by complex mechanisms. To develop more effective therapeutic strategies, it is crucial to understand tumor growth, angiogenesis, and microenvironmental interactions. Although traditional in vivo models such as murine xenografts have long been used to study tumor biology, these approaches are often time-consuming, costly, and ethically constrained. In contrast, the chick embryo chorioallantoic membrane (CAM) assay offers a rapid, cost-effective, and ethically flexible alternative for evaluating tumor development and angiogenesis. This protocol describes an in ovo CAM-based xenograft model in which human breast cancer cells are implanted onto the vascularized CAM of chick embryos. This method enables real-time evaluation of tumor growth. Furthermore, the model allows for manipulation of experimental conditions, including pharmacological treatments or genetic modifications, to study specific molecular mechanisms involved in breast cancer progression. The major advantages of this protocol lie in its simplicity, reduced cost, and capacity for high-throughput screening, making it a valuable tool for translational cancer research.

Congenital renal disorders, such as the Potter sequence, result from renal dysgenesis. To explore a prenatal therapeutic approach for fetuses with kidney insufficiency, we established an in utero transplantation protocol using donor fetal kidneys. Although numerous rodent studies have reported cellular injections into fetal recipients, no protocol to date has described whole-organ transplantation during gestation. Here, we present a step-by-step method for grafting donor fetal kidneys (embryonic day 14.0–16.5) into allogeneic rat fetuses at embryonic day 18.0–18.5, resulting in term neonates that retain the grafts postnatally. A 15–16 G needle preloaded with the donor kidney is inserted transuterinely, depositing the organ into the subcutaneous space of the fetus. Four days later, the term pups are delivered naturally and evaluated for graft development. This protocol enables organ-level transplantation and longitudinal assessment of graft maturation within the unique fetal environment, which differs markedly from adult settings in terms of growth factor availability and immune reactivity. To our knowledge, this is the first protocol to successfully achieve whole-organ transplantation directly into fetuses in utero. Therefore, the model provides a valuable platform for studying developmental organogenesis, fetal immunology, and regenerative strategies that leverage embryonic cues.

The pancreatic islet, the only type of tissue that secretes insulin in response to elevated blood glucose, plays a vital role in diabetes development and treatment. While various islet vascularization strategies have been developed, they have been hindered by major limitations such as relying on pre-patterning and the inability to span long distances. Furthermore, few strategies have demonstrated robust enough vascularization in vivo to support therapeutic subcutaneous islet transplantation. Using adaptive endothelial cells (ECs) reprogrammed by transient expression of the ETS Variant Transcription Factor 2 (ETV-2) gene, we have physiologically vascularized human islets within a generic microchamber and have achieved functional engraftment of human islets in the subcutaneous space of mice. Such adaptive ECs, which we term reprogrammed vascular ECs (R-VECs), have been proven to be a suitable tool for both in vitro disease modeling and in vivo transplantation of not only islets but also other organoids.

Cell transplantation is a promising strategy for treating age-related muscle atrophy, but its critical application remains limited. Cultured myoblasts, unlike freshly isolated muscle stem cells, show poor engraftment efficiency and fail to contribute effectively to muscle regeneration. Moreover, successful engraftment generally requires prior muscle injury, as skeletal muscle regeneration is typically triggered by a damaged microenvironment. These limitations present major obstacles for applying cell therapy to sarcopenia, where muscle degeneration occurs without injury. In this protocol, we describe a novel approach that enables the transplantation of cultured myoblasts into intact skeletal muscle without the need for preexisting injuries or genetic modification. By combining myoblasts with extracellular matrices (ECM), such as Matrigel, which mimic the native muscle niche and support cell survival, adhesion, proliferation, and differentiation, we achieve efficient engraftment and increased muscle mass without the need for preexisting injury. The ECM also provides a scaffold and retains bioactive factors that enhance the regenerative capacity of transplanted cells. This is the first protocol that enables robust myoblast engraftment in non-injury muscle conditions, offering a practical tool for studying and potentially treating sarcopenia.

Adipocytes are endocrine cells that function as the main energy storage in our body. They are commonly used in clinical procedures, including their removal via liposuction and transplantation in plastic surgery. Building on this, adipocytes can be used for ex vivo cellular manipulations, enabling therapeutic modifications that can provide beneficial clinical outcomes after transplantation. Here, we provide a detailed protocol on how to modify adipocytes and adipose organoids using CRISPR activation (CRISPRa), a technology termed adipose manipulation transplantation (AMT).

Humanized immune system (HIS) mice are powerful tools for studying human immune system function and dysfunction and developing human-specific immunotherapeutics. The availability of sophisticated super immunodeficient mouse strains has allowed immune system humanization using transplants of human peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells. HIS mice are used extensively in immune-oncology, while there are fewer studies in autoimmunity, especially multiple sclerosis (MS). Using the protocol described here, we generated HIS mice that show key features of MS not represented in other widely used MS models [1]. Severely immunodeficient NOD.Cg-B2m^em1Tac Prkdc^scid Il2rg^tm1Sug/JicTac (B2m-NOG) mice, which lack murine B, T, and NK cells and murine major histocompatibility class I molecules and have defective innate immune responses, were transplanted with PBMC from HLA-DRB1-genotyped MS patients and healthy donors. Mice were successfully engrafted with hCD4 and hCD8 T and B lymphocytes and developed both spontaneous and experimental autoimmune encephalomyelitis (EAE)-enhanced T-cell lesions in the central nervous system. B-cell engraftment was highest in mice receiving cells from MS patients with serological evidence for Epstein–Barr virus (EBV) reactivation. This humanized MS model shows advantages over EAE, particularly spontaneous hCD8 T-cell lesions in the brain and spinal cord, mixed hCD8/hCD4 T-cell lesions in EAE-immunized mice, and more severe lesions in mice engrafted with PBMC from MS donors carrying the DRB1*15 MS susceptibility allele compared to DRB1*15-positive healthy and DRB1*13-positive MS donors. MS HIS mice represent simple and rapid tools for investigating human immunopathology and the efficacy of therapeutics at a personalized level.

Human immunodeficiency virus (HIV) remains a global health challenge with major research efforts being directed toward the unmet needs for a vaccine and a safe and scalable cure. Antiretroviral therapy (ART) suppresses viral replication but does not cure infection and so requires lifelong adherence. HIV-specific CD8+ T-cell responses play a crucial role in long-term HIV control as demonstrated in elite controllers, highlighting their potential in HIV cure strategies. Various HIV mouse models—including the human-hematopoietic stem cell (Hu-HSC) mouse, the bone marrow, liver, and thymus (BLT) mouse, and the human peripheral blood leukocyte (Hu-PBL) mouse—have deepened the understanding of HIV dynamics and facilitated the development of therapeutics. We developed the HIV participant-derived xenograft (HIV PDX) mouse model to enable long-term in vivo evaluation of bona fide autologous T-cell mechanisms of HIV control, including the antiviral activity of primary memory CD8+ (mCD8+) T cells taken directly from people with or without HIV, as well as testing potential immunotherapies. Additionally, this model faithfully recapitulates virus escape mutations in response to sustained CD8+ T-cell pressure, enabling the assessment of strategies to curb virus escape. In this model, NSG mice are engrafted with purified memory CD4+ (mCD4+) cells and infected with HIV; then, they receive autologous CD8+ T cells or T-cell products. Key advantages of this model include the minimization of graft-versus-host disease (GvHD), which severely limits peripheral blood mononuclear cell (PBMC) or total CD4-engrafted mice, the ability to evaluate long-term natural donor-specific T-cell responses in vivo, and the lack of use of human fetal tissues required for most humanized mouse models of HIV.

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.

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.