Control of osmotic pressure to improve cell viability in cell‐laden tissue engineering constructs

Abstract

Design of tissue engineering strategies deals with the need to balance both biomaterials characteristics and techniques specificities, often resulting in cell-compromising processing conditions. One important factor often disregarded is the osmotic pressure to which cells are exposed. An in-house microfluidic system was used to prove that addition of an osmotic regulator significantly benefits the generation of viable cell-laden hydrogels under harsh processing conditions. Human adipose-derived stem cells were resuspended in 1.5% alginate and 1% gellan gum (GG; w/v) solutions containing different concentrations (0.12 m, 0.25 m and 1.5 m) of sucrose as osmotic regulator. GG (in water) and alginate (in water or phosphate-buffered saline) solutions were used to vary the conditions under which cells were kept prior processing. Independently of the polymer, addition of sucrose did not affect the processing conditions or the viscosity of the solutions, except at 1.5 m. The obtained results clearly demonstrate that inclusion of 0.25 m sucrose during processing of the cell-laden hydrogels allowed to keep cell viability around 80%, in opposition to the 20% observed in its absence, both for GG and alginate-derived hydrogels prepared in water. Impressively, the level of cell viability observed with the inclusion of 0.25 m sucrose, 76% for GG and 86% for alginate, was similar to that obtained with the standard alginate solution prepared in phosphate-buffered saline (82%). The beneficial effect of sucrose was observed within the first 5 min of processing and was maintained for prolonged experimental setups with viability values above 50%, even after a 2-h time-frame and independently of the material.

Phospholipid‐induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration

“…For cell encapsulation, the SF solution was steam‐sterilized at 121°C for 30 min, and DMPG was irradiated by ultraviolet light for 30 min. Then, the lipid film was hydrated with 5% dextrose sterile solution (to control the physiological osmolarity; Carvalho, Gasperini, Ribeiro, Marques, & Reis, ). Afterwards, the suspension was added to a mixture of SF and cells to obtain a final concentration of 3% SF and a cell density of 1 × 10 6 cells per millimetre.…”

Relaxation of Extracellular Matrix Forces Directs Crypt Formation and Architecture in Intestinal Organoids

“…It is possible that at these higher weight percent formulations, an increase in hydrogel swelling (upon equilibration) applies a swelling force to the encapsulated colonies, decreasing their survival . In this case, decreased survival could also be attributed to osmotic effects . While Matrigel might seem to contradict these hypotheses as it is a very soft material, Matrigel and other protein materials have been shown to exhibit strain stiffening behavior, meaning organoids would experience higher local forces …”

Injectable and Magnetic Responsive Hydrogels with Bioinspired Ordered Structures

“…Cell Seeding and Encapsulation in 3D Isotropic and Anisotropic Hydrogels. A 10 wt % gelatin solution was prepared in 0.1X PBS supplemented with 25 mM of sucrose in order to control osmotic pressure and improve cell viability under low ionic strength conditions during processing, as described by Carvalho et al 33 To avoid bacterial growth in biological assays, the nanoparticle suspensions were ultraviolet radiation (UV)-treated for 30 s and dispersed by an ultrasonic processor (40% amplitude, 60 s). Gelatin solution and mTGAse were sterilized using 0.22 μm filters (Biotecnomica, Switzerland), and then an antibiotic/antimicotic solution was added to achieve a final concentration of 1% (v/v).…”

“…All procedures were approved by the University of Minho Ethics Committee. hASCs were routinely isolated by enzymatic digestion, and characterized for stemness potential by flow cytometry andRT-PCR for CD44, STRO-1, CD105 and CD90 markers, as previously reported in Rada, T. et al32 hASCs were maintained in α-MEM supplemented with 10% FBS, and 1% antibiotic/antimicotic solution at 37 ºC, 5% CO2.Cell seeding and encapsulation in 3D isotropic and anisotropic hydrogelsA 10 wt.% gelatin solution was prepared in 0.1X PBS supplemented with 25 mM of sucrose in order to control osmotic pressure and improve cell viability in low ionic strength conditions during processing, as described by Carvalho A., et al33 To avoid bacterial growth in biological assays, the nanoparticles suspensions were ultraviolet radiation (UV) treated during 30 seconds, and dispersed by an ultrasonic processor (40 % amplitude, 60 seconds). Gelatin solution and mTGAse were sterilized using 0.22 m filters (Biotecnomica, Switzerland), and then antibiotic/antimicotic solution was added to achieve a final concentration of 1% (v/v).…”