Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage

Abstract

Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me(2)SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me(2)SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9+/-1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5+/-5.5% and 14.8+/-3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate.

Human dental pulp stem cells derived from different cryopreservation methods of human dental pulp tissues of diseased teeth

“…Recently, Zhurova et al. (16) showed that hDPSCs expressed the gap junction‐forming protein (Connexin‐43) and upon intracellular ice formation retained membrane integrity; however, they lost the ability to proliferate. Nevertheless, for the present study, despite the two different cryopreservation methods having different rates of recovery on primary culture, hDPSCs obtained by these two modalities had similar growth rates (passage 3 sub‐culture) and potency properties.…”

“…It has been shown that water is readily capable of being released and penetrating isolated dental pulp tissues during the development and melting of ice in tissue that has not been entrapped within the inflexible dentin structure of the intact tooth (3,4). Recently, Zhurova et al (16) showed that hDPSCs expressed the gap junctionforming protein (Connexin-43) and upon intracellular ice formation retained membrane integrity; however, they lost the ability to proliferate. Nevertheless, for the present study, despite the two different cryopreservation methods having different rates of recovery on primary culture, hDPSCs obtained by these two modalities had similar growth rates (passage 3 sub-culture) and potency properties.…”

Growth Factor Liberation and DPSC Response Following Dentine Conditioning

“…The sporadic mineralization and random adipogenic differentiation in control cultures on day 21 may be explained by increased cell numbers and cellular contacts as well as paracrine signaling (Tang et al 2010;Zhurova et al 2010). Several cell signaling molecules, including BMPs and insulin growth factor, have been proposed to display pro-osteogenic and proadipogenic effects (James 2013).…”

Role of Cells in Freezing-Induced Cell-Fluid-Matrix Interactions Within Engineered Tissues

“…This ice formation has been thought to be a cause of the damage to the cells and the original ECM microstructure post-thaw. The intracellular ice formation and its impact on cellular viability have been extensively studied as reviewed elsewhere [8–11]. Extracellular ice formation has been examined to quantify the water transport (i.e, cellular dehydration) during freezing and only recently has begun to be studied for its impact on the physiological structure [6, 12–14].…”