Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

The thymus is the central immune organ, within the thymus thymocytes undergo positive and negative selection, and become mature T cells^1,2. Abnormal positive or negative selection results in immunodeficiency or autoimmune pathologies respectively^3,4. 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 selected^6,7. 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 described^8,9. While transplantation protocols have been introduced by several studies^10,11, 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.

Protokół

The presented protocol adheres to the guidelines of the ethics committee of Jinan University regarding animal care.

NOTE: Materials used are listed in the Table of Materials.

1. Isolation of embryonic thymus

1. Autoclave all surgical instruments before the experiment, and sterilize the bench/hood with 70% ethanol.
2. Using carbon dioxide, anesthetize and euthanize the pregnant female mouse (18.5 days post successful mating). Then wipe the abdominal region with 70% ethanol.
   NOTE: Here we mated Insm1^+/lacZ females and Insm1^+/lacZ males. Intraplacental injection of pentobarbital were performed before isolation of embryos from the uterus and decapitation were performed for each embryo.
3. Using scissors, make a "V" shaped cut on the abdomen starting from the bladder and running until each horn of the uterus.
4. Using the scissors, cut the mesometrium and cervix/vagina, and collect the uterus. Place the uterus in a Petri dish containing cold phosphate-buffered saline (PBS) on ice. Next, expose the embryos which are in the enveloped decidua, by cutting the anterior uterine wall from one uterine horn to the other. Using fine tweezers, peel back the enveloped decidua tissues and cut the umbilical cord to release the embryos. Place all the embryos in a new Petri dish on ice until the isolation of the thymus.
5. Wipe an embryo with 70% alcohol and place it in a new Petri dish. From this step on, ensure that sterile conditions are maintained.
6. By cutting close to the lower jaw with scissors, remove the head of the embryo and drain the blood with a paper towel. Next, fix the embryo on the same Petri dish in supine position.
   NOTE: We cut a piece of the tail of each embryo for Insm1 and lacZ genotyping.
7. Using scissors, cut the lateral chest wall horizontally along the axillary front, and then cut the diaphragm to open the chest. The thymus should now be visible as two white lobes located in front of the trachea and adjacent to the heart.
8. Place bent-tip forceps behind the thymus and then pull off the thymus gently. Be sure to check the integrity of the thymus to confirm that it contains two jointed lobes.
9. Wash the thymus with 1x PBS and trim off the connective tissues and blood vessels under a stereomicroscope.

2. Culture of the isolated embryonic thymus

1. Add 500 µL of culture medium (RPMI1640 + 15% fetal bovine serum (FBS) + 100 U/mL penicillin and 100 µg/mL streptomycin) to each well of a 24-well plate. Transfer the clean thymi into the wells with one thymus per well. Figure 1 displays the isolated thymus in culture media.
2. To each thymus-containing well add 2'-deoxygranosine to a final concentration of 1.25 mM.
3. Culture the isolated thymus for eight days, refreshing both the culture media and 2'-deoxygranosine every two days.

3. Establish the subcapsular space in the renal capsule

1. To prepare the needle and the clipped infusion tube (Figure 2), cut the scalp vein needle on the tube part at a 45° angle using scissors.
2. Weigh the nude mouse and then anesthetize it with a pentobarbital (1.5%) injection (75 µg/g body weight).
3. When no reflex following toe pinch is observed, place the mouse on the operating table in a right lateral position.
4. With a 0.5% povidone iodine swab, disinfect the skin twice in the surgical area from the inside to the outside of the body.
5. Using scissors, make a 5–9 mm skin incision parallel to the spine in the left renal area (between the last rib and the iliac crest). Next, open the abdominal cavity by cutting through the subcutaneous tissue and muscle and expose the kidney.
6. With the kidney exposed, use a pair of tweezers in one hand to lift the muscle and fat tissue off the spine-side incision edge. With the other hand, gently squeeze the kidney out (alternatively, the kidney can be squeezed out using fingers of both hands).
7. To ensure that the renal capsule is moist during the surgery, wet the surface of the kidney with saline (0.9% NaCl).
8. Create a nick in the kidney capsule, and gently scratch the renal capsule on the lower right side using the needle tip prepared in step 3.1. The size of the nick should be 1/2–2/3 widths of the kidney; do not scratch on the kidney.
9. Slide the infusion tube prepared in step 3.1 into the nick on the renal capsule. Gently dissociate the renal capsule with the kidney along the kidney's long side until reaching 3–4mm inside the kidney capsule. Draw back the infusion tube; the kidney subcapsular space is established.

4. Transplant the embryonic murine thymus

1. Wash the thymus cultured in step 2.3 twice in saline to deplete the culture media.
2. Connect the clipped infusion tube prepared in step 3.1 to a syringe at its syringe-connecting interface. Aspirate the prepared thymus into the infusion tube slowly.
3. Gently insert the clipped infusion tube into the renal capsule and reach the superior pole. Deliver the thymus into the renal capsule; retract the tube slowly while simultaneously pushing the plunger of the syringe gently.
4. Using an alcohol lamp, slightly heat the needle prepared in step 3.1. After ensuring that the whole thymus is inside the subcapsular space, use the heated needle to cauterize the nick.
5. After cauterization, restore the kidney in the abdominal cavity. Suture the peritoneum and muscle.
6. Using a modified interrupted vertical mattress suture, close the skin incision (tie at least three knots and cut away any excess thread).
7. Using a povidone iodine swab, disinfect the incision. To relieve pain, subcutaneous injection of flunixin (2 µg/g of body weight) was performed during the surgery and then for 3 days after the surgery.
8. Until fully recovered from anesthesia, keep the mouse warm under the infrared lamp.
9. Keep the thymus in the recipient’s kidney capsule for 8 weeks before dissecting the transplanted thymus and conducting phenotypic analysis as previously described^5,8,9.

Wyniki

Here we show the isolated E18.5 thymus containing two complete lobes (Figure 1). Additionally, we show the scalp vein needle that was clipped to form a bevel on the infusion tube (Figure 2). Next, we also show a representative image of the position of the thymus that was transplanted in the renal capsule (Figure 3A) and the thymus after 8 weeks of growth within the recipient mice (Figure 3B). To determi...

Dyskusje

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 transplantation^6,7, our protocol provides a simple alternative procedure on murine embryonic thymus culture and renal subcapsular transplantation for older thymus tissue.

Our protocol...

Materiały

Name	Company	Catalog Number	Comments
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