Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

Abstract

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me 2 SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me 2 SO-and serum-free biopreservation strategies due to safety concerns over Me 2 SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me 2 SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at À 80 � C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me 2 SO-and serumfree cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology

“…Cryomicroscopy was conducted according to a modified protocol that was previously established by our group [63] using an AxioVert M1m microscope (Carl Zeiss, Oberkochen, Germany) with BCS196 Linkam cryostage (Linkam Scientific Instruments, Tadworth, UK) to observe the morphological changes and integrity of the hAM upon two freeze-thaw cycles. The hAMs were cut with a 10 mm puncher, washed twice with PBS (Carl Roth, Karlsruhe, Germany), fixed between two 0.17 mm cover slips, placed onto a quartz crucible (Resultec Analytic Equipment, Illerkirchberg, Germany), and transferred to a temperature-controlled silver block of the cryostage mounted on the microscope.…”

Electrochemical method for isolation of chitinous 3D scaffolds from cultivated Aplysina aerophoba marine demosponge and its biomimetic application

“…Previously, such kinds of 3D chitinous scaffolds isolated from diverse demosponges had been used in tissue engineering [1,3,75,76] and extreme biomimetics [40,77] However, in this study we took the decision to mineralize the electrochemically isolated scaffolds of A. aerophoba origin using mollusk hemolymph as a unique biological system which is responsible for calcification of mechanically damaged shells in vivo.…”

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

“…Cryopreservation with 10% DMSO showed statistically similar survival compared to cryopreservation with 5% DMSO (data not shown). The lower concentration was selected for the further experiments since it is generally preferred to reduce the DMSO concentration in cryopreservation media due to cytotoxicity [ 31 , 32 , 33 , 34 , 35 , 36 , 56 ]. Importantly, it was observed, that even the native (non-cryopreserved) cells harvested from the MK differentiation cultures had no more than 74.25% ± 6.95% of viable cells.…”

“…Among the obligatory requirements to clinically relevant biobanking the key role belongs to the compatibility with good manufacturing practice and good laboratory practice standards, which assumes application of serum- and xeno-free protocols throughout the process. Besides, serious clinical safety concerns are currently being associated with the use of DMSO during cryopreservation [ 31 , 32 , 33 , 34 , 35 , 36 ]. Therefore, these issues were particularly addressed as the core of this study.…”

“…Previously, we developed efficient biobanking technologies for mesenchymal stem cells, blood cells, as well as tissues and tissue engineered constructs [ 26 , 32 , 35 , 63 , 64 ]. Results of these studies show substantial differences in cryopreservation outcomes and necessity to optimize the process for each particular object.…”