Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Summary

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Abstract

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

Introduction

Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing beta cells of the pancreas. The loss of beta cell mass results in insulin deficiency and hyperglycemia. T1D patients rely on multiple daily injections of exogenous insulin and experience episodes of severe hyperglycemia and hypoglycemia throughout their lives. The complications related to these episodes include diabetic retinopathy, decreased kidney function, and neuropathy¹.

Insulin injections are a treatment but not a cure for T1D. Replacing the lost beta cell mass, however, has the potential to reverse the disease by enabling pa....

Protocol

Figure 1: The workflow for transplanting and imaging grafts in an adoptive transfer model of diabetes in mice. NIT-1 cells expressing the firefly transgene luciferase (luc2) are transplanted subcutaneously into NOD-scid mice. The mice are simultaneously injected with autoreactive splenocytes isolated from a spontaneously diabetic NOD mouse. The grafts are imaged at regular intervals by non-invasive bioluminescent imaging. Figure created by BioRen....

Results

An overview of the protocol is outlined in Figure 1. The survival of two cell lines, such as a mutant and a non-targeting control, may be compared, or the survival of one cell line may be measured in multiple groups of mice, such as drug-treated mice versus vehicle-treated controls. Figure 3A shows three 8-week-old female NOD-scid mice transplanted with a non-targeting control (left) and a mutant (right) cell line. The mice were also injected intravenously with .......

Discussion

T1D is a devastating disease for which no cure currently exists. Beta cell replacement therapy offers a promising treatment for patients with this disease, but the critical barrier to this strategy is the potential for recurrent autoimmune attack against the transplanted beta cells. The genetic engineering of SC-beta cells to reduce their immune visibility or susceptibility is one potential solution to this problem. Described here is a protocol for non-invasively imaging transplanted beta cells to measure their survival .......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin, 0.53 mM EDTA	Corning	25-052-CI
293FT	Invitrogen	R70007	Fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen
ACK Lysing Buffer	Gibco	A10492-01
Alcohol prep pads, 70% Isopropyl alcohol	Amazon/Ever Ready First Aid	B08NWF31DX
BD 5ml Syringe Luer-Lok Tip	BD	309646
BD PrecisionGlide Needle 26G x 5/8 (0.45 mm x 16 mm) Sub-Q	BD	305115
BD 1 mL TB Syringe Slip Tip	BD	309659
Blasticidin S HCl	Corning	30-100-RB
Cell strainer premium SureStrain, 70 µm, sterile	Southern Labware	C4070	Or use similar sterile strainer with 40-70um pore size
CellDrop automated cell counter	Denovix	CellDrop BF-PAYG	Or use similar cell counter device
Corning 100 mL Penicillin-Streptomycin Solution, 100x	Corning	30-002-CI
Disposable Aspirating Pipets, Polystyrene, Sterile, Capacity=2 mL	VWR	414004-265	Or use similar aspirating pipette
D-Luciferin, Potassium Salt , Molecular Biology Grade, Powder, >99%	Goldbio	LUCK-100
DMEM, high glucose, pyruvate, no glutamine	Gibco	10313039
Falcon BD tubes, 50 mL	Fisher Scientific	14-959-49A
Fetal Bovine Serum	Gibco	10437-028
Forceps premium for tissues, 1 x 2 teeth 5 in, German Steel	Fisher Scientific	13-820-074
Glucose urine test strip	California Pet Pharmacy	u-tsg100	Or use similar test strip for glucose measurments in urine/blood
GlutaMAX–1 (100x)	Gibco	35050-061
Infrared heating lamp	Cole Parmer	03057-00	Or use similar infrared lamp
Insulin syringe 0.5 mL, U-100 29 G 0.5 in	Becton Dickinson	309306
Isoflurane, USP	Piramal Critical Care	6679401725
IVIS Spectrum in vivo imaging system	Perkin Elmer	124262	Instrument for non-invasively collecting bioluminescent images of transplanted cells
Living Image Analysis Software	Perkin Elmer	128113	Software for collecting and quantifying bioluminescent signal
Microcentrifuge tubes seal-rite, 1.5 mL	USA Scientific	1615-5510	Or use similar sterile microcentrifuge tubes
NIT-1	ATCC	CRL-2055	Pancreatic beta-celll line derived from NOD/Lt mice
NOD.Cg-Prkdcscid/J	The Jackson Laboratory	001303	Mice homozygous for the severe combined immune deficiency spontaneous mutation Prkdcscid, commonly referred to as scid, are characterized by an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment.
NOD/ShiLtJ	The Jackson Laboratory	001976	The NOD/ShiLtJ strain of mice (commonly called NOD) is a polygenic model for autoimmune type 1 diabetes
PBS, pH 7.4	Thermo Fisher Scientific	10010031	No calcium, no magnesium, no phenol red
pCMV-VSV-G	Addgene	8454
pLenti-luciferase-blast	Made in-house	Plasmid available upon request	See Supplemental File 1
pMD2.G	Addgene	12259
pMDLg/pRRE	Addgene	12251
Polyethylenimine, Linear, MW 25,000, Transfection Grade (PEI 25K)	Fisher Scientific	NC1014320
pRSV-Rev	Addgene	12253
Restrainer for rodents, broome-style round 1 in	Fisher Scientific	01-288-32A
Scissors, sharp-pointed	Fisher Scientific	08-940	Or use other scissors made of surgical-grade stainless steel
Tissue-culture treated culture dishes	Millipore Sigma	CLS430167-20EA	Or use other sterile cell culture-treated Petri dishes
Tweezers/Forceps, fine precision medium tipped	Fisher Scientific	12-000-157