We provide a simple and efficient method to transplant 2’-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.
The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2’-deoxyguanosine (to deplete donor’s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient’s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.
The thymus is the central immune organ, within the thymus thymocytes undergo positive and negative selection, and become mature T cells1,2. Abnormal positive or negative selection results in immunodeficiency or autoimmune pathologies respectively3,4. Therefore, thymus organ transplantation is an important approach to study the process of T cells selection in the donor’s thymus. This method is particularly crucial when analyzing thymic epithelial function mediated by gene mutations which cause embryonic lethal phenotype when mutated5.
In order to study the maturation of a recipient’s T cells in the transplanted thymus, depletion of donor’s lymphocytes within the thymus is necessary. For this purpose, embryonic 14-, 15- or 16-day (E14, E15, E16) thymus is usually selected6,7. Thymus from more mature stages can also be successfully depleted of the donor’s lymphocytes by treating with 2’-deoxyguanosine. However, a detailed protocol for depleting lymphocytes and use of older thymus culture has not previously been described8,9. While transplantation protocols have been introduced by several studies10,11, further modification and improvement of these protocols is necessary.
Our protocol is separated into two parts: (i) Depletion of T lymphocytes from late developmental stage E18.5 thymus by culture in 2’-deoxyguanosine-containing media. (ii) Transplantation of the cultured thymus into recipients. In this procedure, we developed a simple way to deliver the large tissue (E18.5 thymus) into the renal capsule with reduced chance of kidney injury. While focussing on later stage thymus, our protocol can also be used directly or with modifications for transplantation of thymus at various developmental stages or other similar sized tissues.
Renal subcapsular transplantation of embryonic thymus is an important method to study the thymic epithelial cells function and the process of T cells maturation in vivo. Although there are several experimental studies on embryonic thymus organ culture and transplantation6,7, our protocol provides a simple alternative procedure on murine embryonic thymus culture and renal subcapsular transplantation for older thymus tissue.
Our protocol…
The authors have nothing to disclose.
This work was supported by the Start Package of Jinan University to S.J. and by Science and Technology Program of Guangzhou China (Grant No. 201704020209 to S.J.). We thank Amy Botta (Department of Biology, York University, Toronto, ON M3J 1P3, Canada) for proofreading and editing of the manuscript.
0.5% Povidone iodine | Shanghai Likang Distinfectant Hi-Tech Co.Ltd | 20171113 | |
0.9% Sodium Chloride Injection | Shandong Qilu Pharmaceuyical Co.Ltd | 2C17112101 | |
1 mL Sterile syringe | Solarbio | YA1090 | |
2’-Deoxyguanosine | MEC | HY-17563 | 1M in DMSO, 1:800 using (final 1.25mM) |
24 Well Plate | Corning Incorporated | Costar 3524 | |
4-0 Surgical suture needles with thread | NingBo Cheng-He Microsurgical Instruments Factory China | YY0166-2002 | |
60mm Cell Culture Dish | Corning Incorporated | 430166 | |
70% ETOH | LIRCON | 20181221 | |
APC anti-mouse CD8a antibodies | Biolegend | 100711 | 1:100 |
Bent-tip fine forceps, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JD1060 | To sterilize before use |
Cefmetazole Sodium for Injection | Sichuan Hexin Pharmaceutical co,Ltd | 17062111 079 | 6mg in 0.5ml 0.9% NaCl solution, 7.5ul/g body weight |
Dissecting scissors, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JC2303 | To sterilize before use |
Fetal bovine serum (FBS) | GIBCO | 10270-106 | |
Fine forceps, JZ 10 cm | Shanghai Medical Devices Group Co.,Ltd. | JD1050 | To sterilize before use |
Flow cytometry | BD | FACSCanto II | |
Flunixin meglumine | MACLIN | F810147 | 1mg in 1ml 0.9% NaCl solution,2ul/g body weight |
Forceps, Dumont#5 | World Precision Instruments | 14098 | To sterilize before use |
Infrared lamp | OTLAN | MT-810 | |
Needle holder, JZ 14 cm | Shanghai Medical Devices Group Co.,Ltd. | J32010 | To sterilize before use |
PE anti-mouse CD4 | Biolegend | 100511 | 1:100 |
Penicillin-Streptomycin mixture | GIBCO | 15140122 | 1:100 |
Pentobarbital sodium salt | Sigma | P3761 | 1.5% solution in PBS, 75ug/g body weight |
RPMI1640 Medium | GIBCO | C14-11875-093 | |
Scalp vein needle | Shanghai Kindly Medical Instruments Co., Ltd | XC001 | |
Spring scissors | VANNAS | S11014-12 | To sterilize before use |
stereomicroscope | OLYMPUS | SZ61 | |
Sterile 15cm cotton swab | Guangzhou Haozheng | 20150014 | |
Sterile gauze 5 cm x 7 cm-8P | Guangzhou Haozheng | 20172640868 | |
Sterile PBS (1x) | GENOM | GNM20012 | |
Tissue forceps, JZ 12.5 cm | Shanghai Medical Devices Group Co.,Ltd. | J41010 | To sterilize before use |