Preserving human cells for regenerative, reproductive, and transfusion medicine

Abstract

Cell cryopreservation maintains cellular life at sub-zero temperatures by slowing down biochemical processes. Various cell types are routinely cryopreserved in modern reproductive, regenerative, and transfusion medicine. Current cell cryopreservation methods involve freezing (slow/rapid) or vitrifying cells in the presence of a cryoprotective agent (CPA). Although these methods are clinically utilized, cryo-injury due to ice crystals, osmotic shock, and CPA toxicity cause loss of cell viability and function. Recent approaches using minimum volume vitrification provide alternatives to the conventional cryopreservation methods. Minimum volume vitrification provides ultra-high cooling and rewarming rates that enable preserving cells without ice crystal formation. Herein, we review recent advances in cell cryopreservation technology and provide examples of techniques that are utilized in oocyte, stem cell, and red blood cell cryopreservation.

Keywords: Cryopreservation; Regenerative medicine; Reproductive medicine; Transfusion medicine; Vitrification.

Figure 1

Minimum volume vitrification methods. (A) An image of closed-pull straw system. (B) Magnified image of straw with oocytes loaded inside. The straw diameter is 200 μm. (C) Image of a viable oocyte just after warming (T1 stage). (D) Image of a viable oocyte 4 hours after warming (T4 stage), just before intracytoplasmic sperm injection. (E) Image of a Day 3 human embryo at 6 cell grade 2 stage. (F) Sonogram showing a ten week clinical pregnancy following a transfer of 3 embryos. A–F are reprinted by copyright permissions from [41]. (G) Cryoloop carrier loaded 4 human oocytes. (H) Cryotop carrier loaded with 5 oocytes. Scale bars indicate 400 μm. G and H are reprinted by copyright permissions from [108].

Figure 2

(A) Surface-based vitrification method. (i) A modification of Thermanox cultivation disc with a small tip to handle with tweezer. (ii) Disc incubation in CPA. Multi photon laser scanning micrographs of hESC-colonies (iii) Control colony, (iv) Cryopreserved colony using slow rate freezing, and (v) Cryopreserved colony using surface based vitrification. Fewer membrane vesicles (arrows) were observed in vitrified sample. Scale bars indicate 100 μm. Reprinted by copyright permissions from [80]. (B) Bulk vitrification method. hESC cell clumps were loaded on the nylon mesh of a cell strainer and incubated in CPA. The inset (right) shows the magnified view of nylon mesh with cell clumps indicated by arrows. Reprinted by copyright permissions from [81].