The Unusual Properties of Polytetrafluoroethylene Enable Massive-Volume Vitrification of Stem Cells with Low-Concentration Cryoprotectants

Abstract

Injectable stem cell-hydrogel constructs hold great potential for regenerative medicine and cell-based therapies. However, their clinical application is still challenging due to their short shelf-life at ambient temperature and the time-consuming fabrication procedure. Banking the constructs at cryogenic temperature may offer the possibility of "off-the-shelf" availability to end-users. However, ice formation during the cryopreservation process may compromise the construct quality and cell viability. Vitrification, cooling biological samples without apparent ice formation, has been explored to resolve the challenge. However, contemporary vitrification methods are limited to very small volume (up to ~0.25 ml) and/or need highly toxic and high concentration (up to ~8 M) of permeable cryoprotectants (pCPAs). Here, we show that polytetrafluoroethylene (PTFE, best known as Teflon for making non-stick cookware) capillary is flexible and unusually stable at a cryogenic temperature. By using the PTFE capillary as a flexible cryopreservation vessel together with alginate hydrogel microencapsulation and Fe₃O₄ nanoparticle-mediated nanowarming to suppress ice formation, massive-volume (10 ml) vitrification of cell-alginate hydrogel constructs with a low concentration (~2.5 M) of pCPA can be achieved. This may greatly facilitate the use of stem cell-based constructs for tissue regeneration and cell based therapies in the clinic.

Keywords: Fe3O4 nanoparticles; PTFE; alginate hydrogel constructs; low-CPA vitrification.

Figure 1.

Controlled generation of core-shell structured alginate hydrogel constructs. A) A schematic illustration of the construct production in a tube-in-tube device. B) Image of the stainless-steel tube-in-tube device with three inlets and one outlet. C) Average size of the constructs generated by the device with three different flow rates of oil. D) Morphology and size distribution of the constructs: (a)-(c) present the merged differential interference contrast (DIC) and fluorescence images showing the core-shell morphology and controllable size of the constructs embedded with fluorescent beads at three different oil flow rates: (a) 150, (b) 300, and (c) 600 μl/min; and (d)–(f) show the size distribution of the constructs produced under the three different flow rates of oil shown in (a)-(c). The core and shell flow rates are the same as that shown in panel C (n = 116–131).

Figure 2.

Characterization of nanoscale, microscale and macroscale materials: A) TEM image of Fe₃O₄ NPs. B) Size distribution of the Fe₃O₄ NPs. C) Zeta potential of the Fe₃O₄ NPs. D) The magnetic property of the NPs. E) The morphology of freeze-dried alginate hydrogel constructs before and after low-CPA vitrification: (a)-(c) the outer surface of constructs before vitrification at different magnifications. (d)-(f) the outer surface of constructs after vitrification with NPs-mediated MIH at different magnifications, and (g)-(i) the inner surface of constructs after vitrification at different magnifications. F) TEM analyses of cells for observing NPs inside cells: TEM images of non-encapsulated (a) and encapsulated cells (b) after 6 hours of co-cultured with Fe₃O₄ NPs. G) Mechanical properties of PTFE capillary: (a)-(b) the morphology of PTFE capillaries before (a) and after (b) cooling and warming repeatedly for 10 times. (c) typical stress-strain curves of PTFE capillary without (W/O) and with (W/) cooling-warming repeatedly for 10 times and (d) the mean values of tensile stress at maximum load W/O and W/ cooling-warming repeatedly for 10 times. Red arrows indicate NPs.

Figure 3.

Vitrification of samples in PPC #1 and PS either without (i.e., in a conventional water bath) and with magnetic induction heating (MIH) for warming. A) Schematic illustration of the procedures for vitrifying samples with PPC #1 either without or with MIH: (a) loading sample into PPC #1, (b) cooling the sample in liquid nitrogen, (c) warming the sample in a 37 ^oC water bath, (d) the non-uniform nature of convective heating in a water bath; (e) magnetic NPs-mediated MIH; and (f) the uniform global and local heating of MIH. B) Images taken during the cooling-warming processes using a plastic straw and PPC #1: (a) conventional heating in 37 °C of water, and (b) NP-mediated MIH.

Figure 4.

Viability and attachment efficiency of the cells released from the constructs, and 3D culture of the intact constructs post-vitrification. A) Viability of MSCs in constructs either W/ or W/O encapsulation (Encap) before and after vitrification with PPC #1 and PS. *: p < 0.05. B) Viability of MSCs W/ or W/O (MIH and Encap) post-vitrification with PPC #1 under different conditions, i.e., 0.1%, 0.5% and 1% (w/v) Fe₃O₄ NPs with MIH under a magnetic field (5, 15, and 25 A). C) Attachment efficiency of MSCs W/ or W/O (MIH and Encap) before and after vitrification with PPC #1. D) DIC and fluorescence images showing the viability of MSCs pre- and post-vitrification using PPC #1 W/ or W/O (Encap and MIH). E) Typical DIC images are showing the attachment efficiency under various conditions. F) Typical DIC and fluorescence images are showing the morphologies of fresh (a) and vitrified (b, with MIH and Encap) constructs and the viability and proliferation of MSCs under 3D culture in the constructs on days 0, 3, 5, and 7.

Figure 5.

Functional properties of MSCs before and after vitrification with MIH and encapsulation. A) Fluorescence immunostaining for CD44 (+), CD90 (+), and CD45 (−) showing the expression of the three markers on MSCs. B) Quantification of CD44 (+), CD90 (+), and CD45 (−) expression on the cells by flow cytometry. C) Qualitative analysis of adipogenic (left) and osteogenic (right) differentiation of MSCs post-vitrification compared to fresh cells. D) Proliferation of MSCs released from constructs post-vitrification compared to fresh cells. E) Typical DIC micrographs between cryopreserved and fresh MSCs showing the cell proliferation.

Figure 6.

Scalability of the PTFE-based technology for vitrification of cell-alginate hydrogel constructs. A) Schematic illustration of PPC #2 and the warming of PPC #2 in electromagnetic coils under alternating current (AC) magnetic field. B) DIC and fluorescence images showing the viability of constructs before and after vitrification using PPC #2 W/ or W/O MIH. C) Viability of constructs W/ or W/O MIH before and after vitrification. D) Typical DIC micrographs of the proliferation of MSCs during three days before and after vitrification with PPC #2. E) Proliferation results of MSCs before and after vitrification.

Figure 7.

The possible mechanisms of low-CPA vitrification developed in this study. A) The fabrication of alginate hydrogel slices. B) Morphological observations of ice formation in either CPA #1 and CPA #2 or in the alginate hydrogel slice without any CPA during cooling and warming. C) A schematic illustration on how alginate hydrogel microencapsulation and MIH might enhance low-CPA vitrification of cells and constructs.