Cell & Animal Models for Immune-Oncology and Cell Therapy

Cyagen+ Cell & Animal Models for Immune-Oncology and CAR-T Cell Therapy


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At present, cell therapy is mostly used in the field of oncology, in which it is necessary to construct stable tumor cell models and animal models to evaluate the therapeutic effect. There are many factors that affect the effectiveness of CAR-T cells, such as transfection efficiency, culture conditions, and cell types. In vitro cell models provide a variety of detection methods for evaluating the effectiveness and specificity of CAR-T therapy. For in vivo evaluation models, it is required that the model have a corresponding immune environment to avoid causing CAR T-cell rejection, and the experimental animals must have a stable living condition. In addition, a mouse model of homologous tumor transplantation can also be used to verify the mechanism and treatment principles of CAR-T.


Cyagen has been committed to the establishment of gene-edited cells and animal models. We have an experienced team of experts and a mature technology platform that provides cell models and animal models for the regulation of target gene expression. For CAR-T and cell therapy research, we have established catalogs with thousands of cell and mouse models for tumor immune research, and provide preclinical in vivo/in vitro pharmacology evaluation services for domestic and foreign pharmaceutical companies, biological companies, scientific research institutes, and hospitals. For example, NKG mice are a severe combined immunodeficiency (SCID) model developed by Cyagen, which are without murine T/B/NK cells and have defective myeloid components. NKG mice can be used to construct various CDX models and reconstruction of the human immune system in a mouse model.


In vitro evaluation models




 Cell Model

Tumor cells expressing CAR-T targets

Luciferase labeled tumor cells

CAR-T cell construction

Overexpression stable transgenic cell Line services


  • Antigen expression is stable: The degree of antigen expression is high, and it still maintains stability after multiple passages.
  • High expression efficiency of CAR molecules: All T cell subtypes have significantly high expression of CAR molecules.
  • One-Stop Services: Multiple verification schemes, complete model construction and identification system.

Case Study

1. Construction of CD19-293T cell line

A 293T cell line with stable expression of CD19 was constructed by lentivirus infection, and the high expression of its CD19 antigen was detected by flow cytometry, shown below.


Figure 1. Flow cytometric detection of CD19 antigen expression in CD19-293T stably transfected cell line.


2. CAR-T cell phenotype analysis and CAR positive rate test results

Monitoring the activated and expanded PBMC, the results showed that high-purity T cells were successfully obtained and which accounted for more than 90% of the cells screened. The subtype analysis results of T cells showed that the proportions of CD4+ and CD8+ cells were 35.93% and 49.45%. Furthermore, FMC63 CAR-T cells were constructed by lentiviral transduction, and FMC63 CAR-T cells were labeled with FMC63 scFv antibodies. The positive rate of FMC63 CAR was 16.99%, indicating that FMC63 CAR-T cells were successfully constructed. 


Figure 2. T cell phenotype analysis and FMC63 CAR-T cell positive rate detection. (A) The proportion of T cells on the tenth day of PBMC activation and expansion and the results of the phenotypic analysis. (B) FMC63 CAR-T cell CAR positive rate test result.


In vivo evaluation models


Type Model Service
Animal model Immunodeficient Mice BALB/c nude mice, NOD scid mice, NKG mice
Humanized immune system mice Hu-PBMC mice, Hu-HSC mice
Cell Line-derived Tumor Xenograft (CDX) model Subcutaneous solid tumor model, In situ solid tumor model, Hematoma model
Homologous tumor mouse model Lung cancer, colorectal cancer, breast cancer, melanoma, and other homologous tumor models
Humanized immune checkpoint mouse model hCTLA-4/hPD-1, hPD-L1mice

1. CAR T Therapy

Chimeric antigen receptor (CAR)-T cell therapy has ushered in a revolutionary era by delivering notably potent and long-lasting clinical outcomes. CARs represent engineered synthetic receptors designed to reprogram lymphocytes, predominantly T cells, to detect and eradicate cells expressing a designated target antigen. CARs bind to specific antigens present on cell surfaces autonomously from the Major Histocompatibility Complex (MHC) receptor, triggering robust T cell activation and robust anti-tumor reactions [1]

NKG mice are a severe immunodeficiency model developed by Cyagen, which are without murine T/B/NK cells and have defective myeloid components. Cyagen has developed a cell line-derived xenograft models (CDX) library, encompassing various solid tumors and hematological system tumors. These resources provide an abundance of services aimed at facilitating research into the effectiveness of CAR-T therapy.

Our CAR-T therapy platform is equipped with a full set of detecting instruments which have allowed us to provide a full range of efficacy services. We can provide a mouse in vivo in situ imaging system, which is utilized to detect various mouse tumor models and metastatic tumor models. And the downstream of various in vitro analyses.

Case study

1.1. CAR T Therapy

NKG mice were inoculated with Nalm6-Luciferase cells via tail vein injection on day 0, followed by CAR-T cell injection on day 3. Tumor load was assessed using in vivo imaging on days 7, 14, 21, and 28. Tumor progression in mice was determined based on the signal intensity of the in vivo imaging.

(A) A CAR-T therapy scheme, the resource from https://medicine.musc.edu/departments/surgery/news-and-announcements/2021/june/car-t-cell-therapy-research;

(B) Experimental flow chart of CD19 CAR-T cell therapy on Nalm6 cell xenograft NKG mouse tumor model; (C) Results of live imaging monitoring of tumor growth, showing that CD19 CAR-T cells can inhibit tumor growth and prolong mouse survival; (D) Mouse survival curve; (E) Mouse weight growth curve.


1. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr; 11(4): 69.

1.2. Antibody Efficacy Evaluation

We evaluated the anti-tumor efficacy of ipilimumab and test articles by implanting MC38 mouse colon cancer cells into B6-hCTLA4 Mice.

(A) Tumor volume inhibition by ipilimumab; (B) Tumor volume inhibition by drugs

2. Categories of Humanized Immune System (HIS) Mice Models

Common Humanized Immune System Mouse Models

Prominent examples of humanized immune system mouse models include humanized PBMC (huPBMC) mice and humanized HSC (huHSC) mice. Cyagen’s NKG immunodeficient mice offer versatility, accommodating transplantation with either PBMCs or HSCs to construct highly effective human immune system mouse models.

This innovative approach facilitates a more accurate simulation of the human immune response, providing exceptional models for preclinical research across various areas, including tumor immunology, autoimmune diseases, and specific infectious diseases.

  • The survival rate of mice is high, and the experimental window is long.
  • Different reconstruction methods of PBMC and HSC can be provided with a high degree of humanization.
  • The model has high uniformity and the data is more convincing for translational research.

Mouse Models Overview

Mouse Name Transplant Graft Source Project Time Immune Reconstitution Status Research Applications
huPBMC-NKG Human peripheral blood mononuclear cells (PBMC) Achieving a humanization ratio of over 40% within 3 weeks. Predominantly T cells, accounting for over 90%. Tumor immunology research; anti-GvHD drug research; infectious disease research; gene therapy; drug target research without cross-reactivity; immunogenicity assays.
huHSC-NKG Human hematopoietic stem cells (HSC) from umbilical cord blood. Reaching a humanization ratio of over 50% within 6 weeks. Reconstruction of various immune cells. Tumor research; immunology research; autoimmune disease research; drug metabolism and toxicity research.
huHSC-NKG-hIL15 Human hematopoietic stem cells (HSC) from umbilical cord blood. Rapid reconstruction achieved in the 3rd week post-transplantation; Reconstruction of various immune cells, especially effective reconstruction of human-derived NK cells; Studies on NK cell development mechanisms, development of NK cell-related tumor immunotherapy, studies on antibody-dependent NK cell-mediated cytotoxicity (ADCC); research on human immune and hematopoietic systems.

Case Study

2.1. Vaccine efficacy evaluation

Our data illustrate the vaccine's ability to elicit tumor volume reduction upon A375 inoculation in huHSC-NKG-ProF mice. We show that the vaccine can increase IFN-γ expression in CD8+ and CD4+ T cells and tetramer in splenocytes.  

(A) A scheme of study design; (B) Tumor volume; (C) IFN-γ% in CD8+ T cells; (D) IFN-γ% in CD4+ T cells; (E) Tetramer in splenocytes

3. Cell line Derived Xenografts (CDX) Model

Cell line-derived xenograft models (CDX models) involve transplanting human-derived tumor cells into mice to evaluate efficacy or conduct tumor-related research in vivo. Cyagen has built an extensive library of human CDX tumor models to facilitate preclinical testing of novel anti-tumor therapeutics, covering diverse cancers such as lung, colon, and bladder cancer. These models meet the needs of various efficacy investigations, as listed in the table.


Cell Line-derived Tumor Xenograft (CDX) models

  • The mechanics of tumor formation among a variety of tumor cells can simulate solid tumors and hematomas.
  • It can effectively support the growth of various tumor cells and CAR-T cells.
  • The CDX model is stable, the experiment is highly reproducible, and the result data is reliable.
  • Highest standards across animal experiment operations and breeding environment.

Case study

3.1. Breast cancer and metastasis model

We inoculated 4T-1-luc cells via carotid artery injection in NKG mice. Brain metastasis signals were detected by day 2.

(A) Study design; (B) In vivo imaging detection; (C) The flux change curve of 4T-luc brain metastasis cancer; (D) Body weight

We inoculated MCF-7-luc cells via mammary fat pad, tibia bone cavity, and iliac branch artery injection to establish a bone metastasis model. On day 19, bone metastasis was observed in the group that received iliac branch artery injection.

(A) Study design; (B) In vivo imaging detection; (C) Bone metastasis imaging detection

3.2. HCC827-LUC human lung adenocarcinoma orthotopic model

We inoculated HCC827-luc cells via left lung lobe injection to establish a lung orthotopic model. On day 12, tumors were observed in the group.

(A) Study design; (B) In vivo imaging detection; (C) Survival rate; (D) Flux change curve; (E) Body weight

3.3.1 MC38 Cancer Syngeneic Model

Subcutaneous transplantation tumor and body weight growth curve of MC38 colorectal cancer cells in mice (n=8). The cells were inoculated subcutaneously into 7-week-old C57BL/6J mice, and tumor volume was measured at different time points. Cell inoculum doses were 1×106/ mouse, 5×105/ mouse, and 1×105/ mouse, respectively. Data are presented as Mean ± SEM. Results indicate that MC38 can be easily modeled in C57BL/6J mice. Tumor volume is expected to reach 100-200 mm^3 by day 6 post-inoculation and 2000 mm^3 by day 19 post-inoculation, which is the experimental endpoint. The therapeutic window is estimated to be around 13 days.

3.3.2 4T1 Cancer Syngeneic Model

Subcutaneous and orthotopic transplantation of 4T1 mouse breast cancer cells and growth curves of tumor volume and body weight (n=10). Cells were inoculated into 7-week-old BALB/C mice via subcutaneous injection and mammary fat pad orthotopic injection, and tumor volumes were measured at various time points. The cell inoculation doses were 1×10^6 per mouse and 5×10^5 per mouse, with data presented as Mean±SEM. Results show that 4T1 is easily modeled in BALB/C mice. Tumor volume is expected to reach 100-200 mm^3 by day 8 post-inoculation and 2000 mm^3 by day 22 post-inoculation, which is the experimental endpoint. The therapeutic window is estimated to be around 14 days.

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