Granted Patent
B2
US 12,471,592 · App. 17/405,735 · Granted Nov 18, 2025
Cytoprotective compositions for short-term cells storage and transportation without cryopreservation and deep freezing
Inventors:
Abhijit Bopardikar (Andheri (West) Mumbai, IN); Padma Priya Anand Baskaran (Mumbai, IN); Sunil Pophale (Mumbai, IN); Andrii Kukharchuk (Mumbai, IN); Oleksandr Kukharchuk (Mumbai, IN); Rohit Kulkarni (Andheri (West) Mumbai, IN)
Assignee:
REELABS PRIVATE LIMITED
View Patent ↗
Loading inventors, assignments & file history…
Abstract
A short-term storage of stem/progenitor cells in regenerative medicine without cryopreservation and cryoprotector. The protective composition of the invention improves the quality and increases the efficiency of cell therapy by keeping stem/progenitor cells free of ultra-low temperatures (liquid nitrogen) and cryoprotectants during the period of their biosafety testing and transportation to a patient's bed.
Claims (8)
1 . An ex vivo suspension of fetal placental mesenchymal stem cells (MSCs), comprising-fetal placental MSCs that express CD73, CD90, and CD105, and a cytoprotective composition,
wherein the cytoprotective composition comprises sodium chloride, potassium chloride, potassium dihydrogen phosphate, potassium a-ketoglutarate, magnesium chloride hexahydrate, histidine-monohydrochloride monohydrate, histidine, tryptophan, mannitol, calcium chloride dihydrate, 3-(2,2,2-trimethylhydrazinium) propionate, quercetin, and taurine;
and wherein the cytoprotective composition preserves the viability and proliferation ability of the fetal placental MSCs for three weeks at 4° C. and one week at room temperature 25° C., and
wherein the ex vivo cell suspension is free of cryoprotectants.
2 . The ex vivo cell suspension according to claim 1 , wherein the cytoprotective composition comprises: 18.0 millimole of the sodium chloride, 15 millimole of the potassium chloride, 1.0 millimole of the potassium a-ketoglutarate, 4.0 millimole of the magnesium chloride hexahydrate, 18.0 millimole of the histidine-monohydrochloride monohydrate, 198 millimole of the histidine, 2.0 millimole of the tryptophan, 30.0 millimole of the mannitol, 0.015 millimole of the calcium chloride dihydrate, 40.0 micromole of the 3-(2,2,2-trimethylhydrazinium) propionate, 20.0 micromole of the quercetin, and 150.0 micromole of the taurine; with a pH of 7.2, and an osmolality of 310 milliosmol/kg.
3 . The ex vivo cell suspension according to claim 1 , wherein the fetal placental MSCs comprises expression of CD73 at 93.5%, expression of CD90 at 93.4%, expression of CD105 at 10.2% after three weeks at 4° C. and one week at 25° C.
4 . The ex vivo cell suspension according to claim 1 , wherein the cryoprotectants include both permeable and non-permeable cryoprotectants.
5 . The ex vivo cell suspension according to claim 1 , wherein the fetal placental MSCs are adapted for regenerative medicine, which includes the treatment of damaged tissues and organs.
Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Nov 3, 2025
From: REELABS PRIVATE LIMITED
To: REELABS, INC.
Reel/Frame 072758/0236 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Sep 3, 2025
From: NAMJOSHI, ZARA
To: REELABS PRIVATE LIMITED
Reel/Frame 072148/0504 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Aug 18, 2021
From: BOPARDIKAR, ABHIJIT; BASKARAN, PADMA PRIYA ANAND; POPHALE, SUNIL; KUKHARCHUK, ANDRII; KUKHARCHUK, OLEKSANDR; KULKARNI, ROHIT
To: REELABS PRIVATE LIMITED, A COMPANY INCORPORATED UNDER PROVISIONS OF THE COMPANIES ACT 1956
Reel/Frame 057218/0583 →
Continuity (1)
References Cited (107)
US 6328960B1
· McIntosh et al.
· 2001
[cited by applicant]
US 6955814B1
· Dobson
· 2005
[cited by applicant]
US 9265795B2
· Hope et al.
· 2016
[cited by applicant]
US 9458424B2
· Comhaire et al.
· 2016
[cited by applicant]
US 20020090369A1
· Murphy
· 2002
[cited by examiner]
US 20090170929A1
· Komeda et al.
· 2009
[cited by applicant]
US 20200397807A1
· Pranesh
· 2020
[cited by examiner]
CN 114377146
· 2022
[cited by examiner]
CN 114377146A
· 2022
[cited by examiner]
WO 1998035551A1
· 1998
[cited by applicant]
WO 2014059316A1
· 2014
[cited by applicant]
Demmy TL et al. Custodiol versus Plegisol: A phase 3 multicentre myocardial protection study. Int J Angiol. 2008 Fall;17(3):149-53. doi: 10.1055/s-0031-1278300. PMID: 22477420; PMCID: PMC2727762 (Year: 2008).
[cited by examiner]
Shelley, Jake. “What is Meldonium/Mildronate?” Published Jan. 4, 2017. https://www.outsideonline.com/health/training-performance/what-meldonium-mildronate/ (Year: 2017).
[cited by examiner]
Kim KM. et al. Assessment of cell viability, early apoptosis, and hematopoietic potential in umbilical cord blood units after storage. Transfusion. Aug. 2015;55(8):2017-22. doi: 10.1111/trf.13120. Epub Apr. 10, 2015. PM…
[cited by examiner]
Yang, J. et al. Natural zwitterionic betaine enables cells to survive ultrarapid cryopreservation. Sci Rep 6, 37458 (2016). https://doi.org/10.1038/srep37458 (Year: 2016).
[cited by examiner]
Dou M. et al. Natural cryoprotectants combinations of I-proline and trehalose for red blood cells cryopreservation. Cryobiology. Dec. 2019;91:23-29. doi: 10.1016/j.cryobiol.2019.11.002. Epub Nov. 3, 2019. PMID: 31693877…
[cited by examiner]
Dambrova M et al. Pharmacological effects of meldonium: Biochemical mechanisms and biomarkers of cardiometabolic activity. Pharmacol Res. Nov. 2016;113(Pt B):771-780. doi: 10.1016/j.phrs.2016.01.019. Epub Feb. 2, 2016. …
[cited by examiner]
Abazari A, Jomha NM, Elliott JA, McGann LE. Cryopreservation of articular cartilage. Cryobiology 2013; 66: 201-209. https://www.sciencedirect.com/science/article/pii/S0011224013001089.
[cited by applicant]
Al-Fageeh MB, Smales CM. Control and regulation of the cellular responses to cold shock: The responses in yeast and mammalian systems. Biochem J 2006; 397: 247-259. https://pubmed.ncbi.nlm.nih.gov/16792527/.
[cited by applicant]
Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Biol Reprod 1994; 50: 103-110. https://pubmed.ncbi.nlm.nih.gov/8312433/.
[cited by applicant]
Arakawa T, Carpenter JF, Yoshiko AK, Crowe JH. The basis of toxicity of certain cryoprotectants: A hypothesis. Cryobiology 1990; 27: 401-415. https://www.researchgate.net/publication/222851017_The_basis_for_toxicity_of_…
[cited by applicant]
Arakawa T, Kita Y, Timasheff SN. Protein precipitation and denaturation by dimethyl sulfoxide.
[cited by applicant]
Aye M, Di Giorgio C, De Mo M, Botta A, Perrin J, Courbiere B. Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: Dimethyl sulfoxide, ethylene glycol and propylene glycol.
[cited by applicant]
Bakaltcheva IB, Odeyale CO, Spargo BJ. Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis.
[cited by applicant]
Banič B, Nipič D, Suput D, Milisav I. DMSO modulates the pathway of apoptosis triggering.
[cited by applicant]
Berthelot-Ricou A, Perrin J, di Giorgio C, de Meo M, Botta A, Courbiere B. Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols.
[cited by applicant]
Bissoyi A, Nayak B, Pramanik K, Sarangi SK. Targeting cryopreservation-induced cell death: A review.
[cited by applicant]
Blake RD, Delcourt SG. Thermodynamic effects of formamide on DNA stability.
[cited by applicant]
Bourne WM, Shearer DR, Nelson LR. Human corneal endothelial tolerance to glycerol, dimethylsulfoxide, 1,2-propanediol, and 2,3-butanediol.
[cited by applicant]
Catchings TT, Beamer WC, Lundy L, Prough DS. Adult respiratory distress syndrome secondary to ethylene glycol ingestion.
[cited by applicant]
Chaytor JL, Tokarew JM, Wu LK, Leclère M, Tam RY, Capicciotti CJ, Guolla L, von Moos E, Findlay CS, Allan DS, Ben RN. Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.
[cited by applicant]
Chen SL, Tian YS. Cryopreservation of flounder (
[cited by applicant]
Ciotti PM, Porcu E, Notarangelo L, Magrini O, Bazzocchi A, Venturoli S. Meiotic spindle recovery is faster in vitrification of human oocytes compared to slow freezing.
[cited by applicant]
Clark P, Fahy GM, Karow AM., Jr. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices.
[cited by applicant]
Damien M, Luciano AA, Peluso JJ. Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change.
[cited by applicant]
De Ménorval MA, Mir LM, Fernández ML, Reigada R. Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: A comparative study of experiments in silico and with cells.
[cited by applicant]
Elmoazzen HY, Poovadan A, Law GK, Elliott JA, McGann LE, Jomha NM. Dimethyl sulfoxide toxicity kinetics in intact articular cartilage.
[cited by applicant]
Eroglu A, Bailey SE, Toner M, Toth TL. Successful cryopreservation of mouse oocytes by using low concentrations of trehalose and dimethylsulfoxide.
[cited by applicant]
Eroglu A, Toner M, Toth TL. Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes.
[cited by applicant]
Fahy GM, Levy DI, Ali SE. Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions.
[cited by applicant]
Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT. Cryoprotectant toxicity and cryoprotectant toxicity reduction: In search of molecular mechanisms.
[cited by applicant]
Fahy GM, Wowk B, Wu J, Paynter S. Improved vitrification solutions based on the predictability of vitrification solution toxicity.
[cited by applicant]
Fahy GM, Wowk B, Wu J. Cryopreservation of complex systems: The missing link in the regenerative medicine supply chain.
[cited by applicant]
Fahy GM. Cryoprotectant toxicity neutralization.
[cited by applicant]
Fahy GM. Cryoprotectant toxicity reduction: Specific or non-specific?
[cited by applicant]
Fahy GM. Cryoprotectant toxicity: Biochemical or osmotic?
[cited by applicant]
Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology.
[cited by applicant]
Galvao J, Davis B, Tilley M, Normando E, Duchen MR, Cordeiro MF. Unexpected low-dose toxicity of the universal solvent DMSO.
[cited by applicant]
Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe M, Larman MG. Analysis of oocyte physiology to improve cryopreservation procedures.
[cited by applicant]
Gardner TB, Manning HL, Beelen AP, Cimis RJ, Cates JM, Lewis LD. Ethylene glycol toxicity associated with ischemia, perforation, and colonic oxalate crystal deposition.
[cited by applicant]
Gautam SK, Verma V, Palta P, Chauhan MS, Manik RS. Effect of type of cryoprotectant on morphology and developmental competence of in vitro-matured buffalo (
[cited by applicant]
Gupta MK, Uhm SJ, Lee HT. Effect of vitrification and beta-mercaptoethanol on reactive oxygen species activity and in vitro development of oocytes vitrified before or after in vitro fertilization.
[cited by applicant]
Gurtovenko AA, Anwar J. Modulating the structure and properties of cell membranes: The molecular mechanism of action of dimethyl sulfoxide.
[cited by applicant]
Hakura A, Mochida H, Yamatsu K. Dimethyl sulfoxide (DMSO) is mutagenic for bacterial mutagenicity tester strains.
[cited by applicant]
Hanslick JL, Lau K, Noguchi KK, Olney JW, Zorumski CF, Mennerick S, Farber NB. Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system.
[cited by applicant]
Hess R, Bartels MJ, Pottenger LH. Ethylene glycol: An estimate of tolerable levels of exposure based on a review of animal and human data.
[cited by applicant]
Homsi E, Janino P, de Faria JB. Role of caspases on cell death, inflammation, and cell cycle in glycerol-induced acute renal failure.
[cited by applicant]
Ivanov IT. Rapid method for comparing the cytotoxicity of organic solvents and their ability to destabilize proteins of the erythrocyte membrane.
[cited by applicant]
Jomha NM, Law GK, Abazari A, Rekieh K, Elliott JA, McGann LE. Permeation of several cryoprotectant agents into porcine articular cartilage.
[cited by applicant]
Jomha NM, Weiss AD, Fraser Forbes J, Law GK, Elliott JA, McGann LE. Cryoprotectant agent toxicity in porcine articular chondrocytes.
[cited by applicant]
Korrapati MC, Shaner BE, Schnellmann RG. Recovery from glycerol-induced acute kidney injury is accelerated by suramin.
[cited by applicant]
Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes.
[cited by applicant]
Lawson A, Ahmad H, Sambanis A. Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions.
[cited by applicant]
Liu J, Yoshikawa H, Nakajima Y, Tasaka K. Involvement of mitochondrial permeability transition and caspase-9 activation in dimethyl sulfoxide-induced apoptosis of EL-4 lymphoma cells.
[cited by applicant]
Lubzens E, Tamar G, Pekarsky I, Blais I, Chapovetsky V, Admon A. Proteomic analysis on the effect of cryopreservation procedures on fish oocytes (abstract 73).
[cited by applicant]
Macías García B, Ortega Ferrusola C, Aparicio IM, Miró-Morán A, Morillo Rodriguez A, Gallardo Bolaños JM, González Fernández L, Balao da Silva CM, Rodríguez Martínez H, Tapia JA, Peña FJ. Toxicity of glycerol for the st…
[cited by applicant]
Markarian SA, Bonora S, Bagramyan KA, Arakelyan VB. Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on
[cited by applicant]
Meryman HT. Cryopreservation of living cells: Principles and practice.
[cited by applicant]
Mitrus I, Smagur A, Giebel S, Gliwinska J, Prokop, Glowala-Kosinska M, Chwieduk A, Sadus-Wojciechowska M, Tukiendorf A, Holowiecki J. A faster reconstitution of hematopoiesis after autologous transplantation of hematopo…
[cited by applicant]
Morley P, Whitfield JF. The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types.
[cited by applicant]
Mullen SF, Li M, Li Y, Chen ZJ, Critser JK. Human oocyte vitrification: The permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification.
[cited by applicant]
Neild DM, Brouwers JF, Colenbrander B, Agüero A, Gadella BM. Lipid peroxide formation in relation to membrane stability of fresh and frozen thawed stallion spermatozoa.
[cited by applicant]
Neutelings T, Lambert CA, Nusgens BV, Colige AC. Effects of mild cold shock (25° C.) followed by warming up at 37° C. on the cellular stress response.
[cited by applicant]
Notman R, den Otter WK, Noro MG, Briels WJ, Anwar J. The permeability enhancing mechanism of DMSO in ceramide bilayers simulated by molecular dynamics.
[cited by applicant]
O'Connell M, McClure N, Lewis SE. The effects of cryopreservation on sperm morphology, motility and mitochondrial function.
[cited by applicant]
Ogura T, Shuba LM, McDonald TF. Action potentials, ionic currents and cell water in guinea pig ventricular preparations exposed to dimethyl sulfoxide.
[cited by applicant]
Pegg DE. Principles of cryopreservation.
[cited by applicant]
Porter WH. Ethylene glycol poisoning: Quintessential clinical toxicology; analytical conundrum.
[cited by applicant]
Qi W, Ding D, Salvi RJ. Cytotoxic effects of dimethyl sulphoxide (DMSO) on cochlear organotypic cultures.
[cited by applicant]
Ren L, Zhang D, Jiang XN, Gai Y, Wang WM, Reed BM, Shen XH. Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in
[cited by applicant]
Richardi J, Krienke H, Fries HF. Dielectric constants of liquid formamide, N-methylformamide and dimethylformamide via molecular Ornstein-Zernike theory.
[cited by applicant]
Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: A 1996 review.
[cited by applicant]
Samoszuk M, Reid ME, Toy PT. Intravenous dimethylsulfoxide therapy causes severe hemolysis mimicking a hemolytic transfusion reaction.
[cited by applicant]
Shlafer M, Karow AM., Jr. Pharmacological effects of dimethyl sulfoxide on the mammalian myocardium.
[cited by applicant]
Somfai T, Nakai M, Tanihara F, Noguchi J, Kaneko H, Kashiwazaki N, Egerszegi I, Nagai T, Kikuchi K. Comparison of ethylene glycol and propylene glycol for the vitrification of immature porcine oocytes.
[cited by applicant]
Songsasen N, Yu IJ, Ratterree MS, VandeVoort CA, Leibo SP. Effect of chilling on the organization of tubulin and chromosomes in rhesus monkey oocytes.
[cited by applicant]
Stroh C, Cassens U, Samraj A, Sibrowski W, Schulze-Osthoff K, Los M. The role of caspases in cryoinjury: Caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells.
[cited by applicant]
Sutton RL. Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides.
[cited by applicant]
Szurek EA, Eroglu A. Comparison and avoidance of toxicity of penetrating cryoprotectants.
[cited by applicant]
Taylor MJ, Baicu S. Review of vitreous islet cryopreservation: Some practical issues and their resolution.
[cited by applicant]
Valdez CA, Abas Mazni O, Takahashi Y, Fujikawa S, Kanagawa H. Successful cryopreservation of mouse blastocysts using a new vitrification solution.
[cited by applicant]
Wang X, Hua TC, Sun DW, Liu B, Yang G, Cao Y. Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability.
[cited by applicant]
Wang Y, Tao F, Xu P. Glycerol dehydrogenase plays a dual role in glycerol metabolism and 2,3-butanediol formation in Klebsiella pneumoniae.
[cited by applicant]
Warheit DB, Kinney LA, Carakostas MC, Ross PE. Inhalation toxicity study of formamide in rats.
[cited by applicant]
Weber MH, Marahiel MA. Coping with the cold: The cold shock response in the Gram-positive soil bacterium
[cited by applicant]
Westh P. Preferential interaction of dimethyl sulfoxide and phosphatidyl choline membranes.
[cited by applicant]
Wood DC, Wirth NV, Weber FS, Palmquist MA. Mechanism considerations of dimethyl sulfoxide (DMSO)-lenticular changes in rabbits.
[cited by applicant]
Wusteman MC, Pegg DE, Robinson MP, Wang LH, Fitch P. Vitrification media: Toxicity, permeability, and dielectric properties.
[cited by applicant]
Yu ZW, Quinn PJ. Dimethyl sulphoxide: A review of its applications in cell biology.
[cited by applicant]
Zhang YZ, Zhang SC, Liu XZ, Xu YJ, Hu JH, Xu YY, Li J, Chen SL. Toxicity and protective efficiency of cryoprotectants to flounder (
[cited by applicant]