1. Introduction
In 2017, Liu Xuefeng's research group from Georgetown University in the United States proposed a technique for unrestricted cultivation of normal and tumor cells using Conditional Reprogrammed Cells (CRCs) in the laboratory. This technology refers to co culturing Swiss-3T3-J2 mouse fibroblast cells irradiated with cesium source gamma rays with human normal cells or tumor cells. By adding the Rho associated kinase (ROCK) inhibitor Y-27632, normal cells or tumor cells can obtain partial stem cell characteristics and unlimited replication ability in vitro.
2. Technical Process [1]
2.1 Cell culture in the feeding layer
(1) Cultivate Swiss-3T3-J2 mouse fibroblasts in a 37 ° C, 5% CO2 incubator and passage 2-3 times per week.
(2) During passage, rinse once with PBS and incubate at room temperature for 20 seconds with 0.05% trypsin/EDTA solution. Add 4 times the volume of DMEM complete culture medium to terminate digestion.
(3) 300g, centrifuge for 5 minutes.
(4) The cell pellet was resuspended in 10ml of DMEM complete culture medium and seeded with 1x103 cells/cm2 cells. It was then cultured in a 37 ° C, 5% CO2 incubator.
2.2 Transportation and Handling of Fresh Organizations
(1) Fresh tissue is transported and processed under sterile conditions within 12 hours after surgery.
(2) Quickly rinse the excised tissue samples with 95-100% ethanol (up to 3 seconds), then wash them with cold (4 ℃) PBS and transfer them to sterile culture dishes.
(3) Use tweezers and a surgical knife to remove residual adipose tissue.
(4) Cut the tissue into small pieces smaller than 10mm. Place the small tissue pieces in 5ml of tissue protective solution and transport them to the laboratory via cold chain (2-8 ℃)
2.3 Primary cell isolation
(1) Prepare 10x collagenase/hyaluronidase solution in a 15ml centrifuge tube. Mix F medium and 10x collagenase/hyaluronidase mixed solution in a ratio of 3:1.
(2) Quickly rinse the excised tissue samples with 95-100% ethanol (up to 3 seconds), then rinse with cold (4 ° C) PBS.
(3) Cut the organization into small pieces smaller than 1mm3.
(4) Transfer the tissue fragments to a 15ml centrifuge tube containing 4ml of culture medium/collagenase/hyaluronidase/distribution enzyme, and shake at 37 ° C for 1-3 hours.
(5) After dissociation, centrifuge at 500g for 5 minutes at 4 ° C and discard the supernatant. Resuspend the cell pellet in 10 ml of complete DMEM culture medium.
(6) Filter the cell suspension into a new 50ml centrifuge tube using a 100 µ m cell filter. Centrifuge at 300g for 5 minutes at 4 ℃, discard the supernatant.
2.4 Radiation and Co culture of Feeding Layer Cells
(1) Take Swiss-3T3-J2 mouse fibroblasts cultured to 80-90% confluence degree, wash once with PBS, and then incubate at room temperature with 0.05% trypsin/EDTA solution for 20 seconds.
(2) Add 4 times the volume of DMEM complete culture medium to terminate digestion.
(3) 300g, centrifuge for 5 minutes. The cell pellet was resuspended in 10ml of DMEM complete culture medium and subjected to appropriate irradiation at 30Gy (parallel culture was used to ensure that J2 cells did not proliferate after irradiation).
(4) Immediately after irradiation, J2 cells were placed in T25 flasks (1 × 104 cells/cm2) along with treated tissue or desired epithelial cells, and placed in complete F medium.
(5) Maintain co culture at 37 ° C, 95% humidity, and 5% CO2 incubator until confluence.
2.5 Differential trypsin treatment co cultured cells for passage
(1) Take co cultured and confluent cells, wash once with PBS, then incubate at room temperature with 0.05% trypsin/EDTA solution for 20 seconds, observe digestion under a microscope, and only maintain the presence of epithelial cell clones.
(2) Wash the epithelial cells with PBS, then reprocess with 1ml of 0.05% trypsin/EDTA at 37 ℃ for 5 minutes.
(3) Add 4 times the volume of complete DMEM culture medium to terminate cell digestion, and collect the mixture in a 15ml centrifuge tube.
(4) Centrifuge at 300g for 5 minutes at 4 ℃, discard the supernatant. Cell precipitation was resuspended in F medium and passaged in a certain proportion.
3. Application
CR technology has brought hope to the research of cell immortalization and has shown great application prospects in fields such as primary cell culture and tumor therapy.
3.1 Applications in primary cell culture [2]
At present, CR technology has been used to isolate and culture bladder cancer cells exfoliated from the lung, breast, colon, esophagus, prepuce epithelium, cervix, vagina and urine [3-5]. Meanwhile, this technology cultivates cells and exhibits stem cell characteristics in adult epithelial cells [6].
3.2 Application in tumor treatment [7]
Yuan et al. [8] established normal and tumor lung tissue cell cultures of patients with invasive recurrent respiratory papillomas using CRCs. This study applied genomics analysis to discover that laryngeal cancer tumor cells contain the wild-type 7.9-kb human papillomavirus type 11 genome, while lung tumor cells contain the 10.4-kb genome. Further benefits
Using drug sensitivity testing, it was determined that vorinostat was a specific potential therapeutic agent. After 3 months of clinical treatment, the lesion began to shrink.
Timofeeva et al. [5] used cultured normal prostate cells and tumor cells to demonstrate that basal cell populations are the main source of prostate tumors. They also demonstrated that CRCs can maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and Y-27632, and can fully differentiate once placed in vivo or under conditions that simulate their natural environment. Researchers studied the sensitivity of docetaxel to CRCs, and the IC50 values of matched normal and tumor cells were 1.79 µ m and 0.29 µ m, respectively. These data suggest that matched normal and tumor cells can be used for personalized treatment of patients or for the discovery of novel drugs.
Li Zhangyan et al. [10] established an in vitro culture system for lung cancer using CRCs technology, and used MTS method to detect the sensitivity of 6 lung adenocarcinoma cells to conventional chemotherapy drugs such as cisplatin, carboplatin, nedaplatin, and vinorelbine. The results showed that the overall sensitivity of lung adenocarcinoma cells to platinum based drugs was high, while the sensitivity to vinorelbine was low, which is consistent with clinical conditions and similar to previous research results.
4. References
【1】Liu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A, Simic V, Zheng YL, Sripadhan P, Chen C, Lu J, Hou TW, Choudhury S, Kallakury B, Tang DG, Darling T, Thangapazham R, Timofeeva O, Dritschilo A, Randell SH, Albanese C, Agarwal S, Schlegel R. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc. 2017 Feb;12(2):439-451. doi: 10.1038/nprot.2016.174. Epub 2017 Jan 26. PMID: 28125105; PMCID: PMC6195120.
【2】Ye Fangdie, Jiang Haowen Progress in the application of conditional reprogramming culture technology for long-term isolation and cultivation of primary cells in vitro [J] Fudan Journal: Medical Edition, 2020, 47 (5): 7. DOI: 10.3969/j.issn.1672-8467.2020.05.019
【3】Ahmad,M,Alamri,et al.Primary cancer cell culture: mammary-optimized vs conditional reprogramming[J].Endocrine Related Cancer, 2016.
【4】Jensen T J , Foster C , Sayej W ,et al.Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation[J].Journal of Visualized Experiments Jove,
【5】Timofeeva O A , Palechor-Ceron N , Li G ,et al.Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer[J].Oncotarget, 2016.
【6】Suprynowicz F A , Kamonjoh C M , Ewa K ,et al.Conditional cell reprogramming involves non-canonical β-catenin activation and mTOR-mediated inactivation of Akt[J].Plos One, 2017, 12(7):e0180897.
【7】Zhang Huihui, Yu Wanjun Research progress of conditionally reprogrammed cells in tumor therapy [J] Chinese Clinical Oncology, 2018, 45 (15): 4. DOI: CNKI: SUN: ZGZL. 0.2018-15-015
【8】Use of reprogrammed cells to identify therapy for respiratory papillomatosis. [J].New England Journal of Medicine, 2012, 367(13):1220.
【9】Li Zhangyan, Tang Yubo, Luo Honghe, etc Long term culture and personalized drug sensitivity study of human primary lung cancer cells in vitro [J] Today's Pharmacy, 2017, 27 (2): 6. DOI: CNKI: SUN: YAXU. February 2013