Development of a Cryopreservation Technique for Xenogeneic Kidney Grafts: Evaluation Using a Mouse Model

Abstract

To align the xeno-metanephros and renal progenitor cell timing for transplantation treatments, cryopreservation techniques and an efficient transportation of regenerated renal products such as xeno-metanephroi and renal progenitor cells should be established. Therefore, we propose a novel method of xenogeneic regenerative medicine for patients with chronic kidney disease by grafting porcine fetal kidneys injected with human renal progenitor cells. To develop a useful cryopreserve system of porcine fetal kidney and human renal progenitor cells, we examined the cryopreservation of a fetal kidney implanted with renal progenitor cells in a mouse model. First, we developed a new method for direct cell injection under the capsule of the metanephros using gelatin as a support for unzipped fetal kidneys. Then, we confirmed in vitro that the nephrons derived from the transplanted cells were regenerated even after cryopreservation before and after cell transplantation. Furthermore, the cryopreserved chimeric metanephroi grew, and regenerated nephrons were observed in NOD. We confirmed that even in cryopreserved chimeric metanephroi, transplanted cell-derived nephrons as well as fresh transplants grew.

Cryopreservation of Fetal Porcine Kidneys for Xenogeneic Regenerative Medicine

“…MNBs were cryopreserved via vitrification as previously reported [ 9 , 10 , 12 ]. First, the MNBs were equilibrated in base medium (MEM α supplemented with 20% fetal bovine serum (FBS; SH30070.03, HyClone Laboratories, Inc., Logan, UT, USA) and 1% antibiotic–antimycotic solution [15,240,062; Thermo Fisher Scientific, Waltham, MA, USA]) with 7.5% ethylene glycol (EG; 055-00996; Wako, Osaka, Japan) and 7.5% dimethyl sulfoxide (DMSO; 317275-100ML; Millipore, Burlington, MA, USA) on ice for 15 min and soaked in base medium with 15% EG and 15% DMSO on ice for an additional 15 min.…”

“…For clinical application, properly preserved, ready-to-use grafts may be useful because the timing of transplantation must be adjusted according to the patient’s condition. We have previously demonstrated that cryopreserved fetal pig [ 9 ] and mouse [ 10 ] MNs differentiated well. However, an effective method to evaluate vitrified MNs has not been investigated thus far.…”