Osmotic stresses during cryoprotectant loading induce changes in cellular volume, leading to membrane damage or even cell death. Appropriate model-guided mitigation of these osmotic gradients during cryoprotectant loading is currently lacking, but would be highly beneficial in reducing viability loss during the loading process. To address this need, we reformulate the two-parameter formalism described by Jacobs and Stewart for cryoprotectant loading under the constraint of constant cell volume. We then derive simple, concise, analytic solutions to these equations, showing the transient extracellular permeating and nonpermeating cryoprotectant concentrations required to load a cell at constant volume, thus eliminating osmotic stresses during cryoprotectant loading. Additionally, we show analytic approximations of both ramp (linear) as well as step-wise loading and how one can use the hydraulic conductivity L_p, membrane permeability P_s, cell volume V_o, and osmotically inactive fraction to derive cryoprotectant loading protocols that minimize osmotic stress. We also present timescales for water and cryoprotectant transport which can be used to estimate loading times as well as L_p and P_s. We discuss how previous optimized loading strategies are inherently sensitive to parameter uncertainties and biological variability, increasing the likelihood of exceeding critical osmotic limits. By contrast, the proposed protocol provides a larger buffer against deviations, offering a safer and more robust solution to CPA loading. Importantly, we demonstrate that the volume-loss-free CPA loading protocols outlined in this paper occur on the same timescale as conventional and step-loading methods, suggesting that these protocols could be a safer, more efficient alternative for CPA loading.

01 Mar 2025·Cryobiology

An efficient combination of a CPA loading protocol, metal container, and storage method for successful articular cartilage vitrification

Article

Author: Shahsavari, Mohammadhamed ; Ribeiro Rodrigues, Ana Paula ; Elliott, Janet A W ; Jomha, Nadr M

Successful cryopreservation of articular cartilage (AC) depends on a number of variables, including heat transfer, sample packaging for cryogenic storage, cryoprotectant agent (CPA) concentration (toxicity), and CPA permeation into AC. In the first experiment of the present study, we used a combination of our established vitrification protocol (430-min multi-step loading Protocol 8) and the metal Ovarian Tissue Cryosystem (OTC) as a closed vitrification-storage-rewarming container to vitrify 7-mm diameter porcine osteochondral dowels with 2 methods of storage (storage in the OTC with or without a surrounding vitrification solution). In the second experiment, in an attempt to introduce a more reproducible and safe vitrification protocol, we employed our successful Protocol 8 with the OTC as the CPA loading container and a cryobag as the storage container (with or without surrounding vitrification solution). In both experiments, post-warming normalized chondrocyte viabilities were assessed. The results of the first experiment demonstrated that a combination of the established step-wise CPA loading-vitrification-rewarming protocol (Protocol 8) with the metal OTC as the only container through all steps of CPA loading, vitrification, and rewarming produced a positive result. In the second experiment, combination of Protocol 8, the OTC as the CPA loading system and a cryobag as the storage container in both vitrified groups with or without solution resulted in high normalized chondrocyte viabilities (94.63 % and 96.17 %, respectively) post vitrification in 7-mm diameter porcine osteochondral dowels. Overall, this study demonstrated that by employing our established Protocol 8, using the metal OTC as a CPA loading container and a cryobag as the storage method we could achieve a reproducible method for clinical applications involving vitrified articular cartilage.

01 Mar 2025·Cryobiology

Detecting changes of testicular interstitial cell membranes with a fluorescent probe after incubation and cryopreservation with cryoprotective agents

Article

Author: Pakhomov, Oleksandr ; Posokhov, Yevgen

Membrane alterations are among central factors predetermining cell survival during cryopreservation. In the present research, we tested some serum-/xeno-free cryoprotective compositions including dimethyl sulfoxide (Me₂SO) and polymers for their osmotic impact and toxicity towards testicular interstitial cells (ICs). IC survival was determined after their contact with Me₂SO, dextran (D40), hydroxyethyl starch (HES), polyethylene glycols (PEG1500 and PEG400), or after cryopreservation and cryoprotective agent (CPA) removal. A ratiometric fluorescent membrane probe 2-(2'-hydroxyphenyl)-5-phenyl-1,3-oxazole (probe O1O) was applied to assess changes in the plasma membrane of ICs. The cell survival study has shown that Me₂SO decreased IC survival in a time- and dosage-dependent manner. The CPA decreased the metabolic activity of ICs, thus implying its toxic effect on the living cell as a whole. Using probe O1O, we have demonstrated that the toxic effect also influenced the plasma membrane. IC membranes were not altered after incubation with 0.7 M Me₂SO. The presence of D40, HES, or PEGs in such Me₂SO containing media resulted in plasma membrane hydration and damage to the membranes of cells incubated with PEGs. Cryopreservation caused pronounced membrane dehydration of the survived ICs even after CPA removal in PEG-containing media and low indicators of IC survival. Interestingly, cryopreservation with the best cryoprotective media supplemented with 0.7 M Me₂SO and 100 mg/ml D40 resulted in minimal membrane alterations, thus implying its higher ability to protect membranes during cryopreservation.

100 Deals associated with Alaptide

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Indication	Highest Phase	Country/Location	Organization	Date
Alzheimer Disease	Phase 3	Czechia	-	-
Duodenal Ulcer	Phase 3	Czechia	-	-
Parkinson Disease	Phase 3	Czechia	-	-
Skin Diseases	Phase 3	-	Research Institute for Pharmacy and Biochemistry	-
Skin Diseases	Phase 3	Czechia	-	-

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Study	Phase	Population	Analyzed Enrollment	Group	Results	Evaluation	Publication Date


Tandem Meetings ASTCT and CIBMTR2016	Not Applicable	Stem cell mobilisation	-	G-CSF alone	ykgajtnqbf(ibvdkaxozo) = yfupbmiiet ekbxicqlwz (udzsmyhptf, 0.5 - 9.8)	Positive	01 Mar 2016
				CPA + G-CSF	ykgajtnqbf(ibvdkaxozo) = dhjilnopnl ekbxicqlwz (udzsmyhptf, 0.5 - 19)
SITC2013	Phase 3	MUC1 +	-	Cyclophosphamide (CPA)	teuyjxqubx(kxzjhibiuh) = higher pvimzectrh (zsgtodvbzl ) View more	Positive	07 Nov 2013
				Tecemotide

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