# US 11,297,828 B2 · Surface tension mediated lyo-processing technique for preservation of biologics > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,297,828 B2 - Document ID: US11297828B2 - Status: Granted - Kind: B2 - Application number: 16/085,379 - Filed: 2017-03-14 - Published: 2019-03-21 - Granted: 2022-04-12 - Earliest claimed: 2016-03-14 - Assignees: The Regents of the University of Michigan - Applicants: The Regents of the University of Michigan - Inventors: Nilay Chakraborty - Web page: https://patentgrep.com/patent/US11297828B2 ## CPC - A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators - A01N 1/128: Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking - A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time - A01N 1/165: Pressure processes, e.g. following predefined pressure changes over time - F26B 5/06: the process involving freezing - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - F26B: DRYING SOLID MATERIALS OR OBJECTS BY REMOVING LIQUID THEREFROM (drying devices for combines A01D41/133; racks for drying fruit or vegetables A01F25/12; drying foodstuffs A23; drying hair A45D20/00; body-drying implements A47K10/00; drying household articles A47L; drying gases or vapours B01D; chemical or physical processes for dewatering or like separating liquids from solids B01D43/00; centrifugal apparatus B04; drying ceramics C04B33/30; drying yarns or fabrics in association with some other form of treatment D06C; drying frames for laundry without heating or positive air circulation, domestic laundry-or spin-driers, wringing or hot pressing laundry D06F; furnaces, kilns, ovens F27) ## Abstract Methods and devices for lyo-processing biological materials are provided. The methods include submerging the biological molecules or cells in a buffer solution comprising trehalose, withdrawing the biological molecules or cells from the buffer solution in an environment that does not include oxygen to generate lyo-stabilized biological molecules or cells, and storing the lyo-stabilized biological molecules or cells. The devices include a motor that lowers a horizontal member having a clamp for receiving a substrate toward a solvent reservoir and raises the horizontal member having the clamp up and away from the solvent reservoir. ## Claims ### Claim 1 A method for lyo-processing a biological material, the method comprising: generating a layer of the biological material on a substrate; submerging the substrate in a lyo-processing solution comprising a lyoprotectant; raising the substrate out of the lyo-processing solution; and contacting the biological material with an inert gas as the substrate is raised out of the lyo-processing solution to generate a lyo-stabilized biological material on the substrate. ### Claim 2 (depends on 1) The method according to claim 1, wherein the generating a layer of the biological material on a substrate comprises generating a cell monolayer on a substrate. ### Claim 3 (depends on 2) The method according to claim 2, wherein the generating a cell monolayer on a substrate comprises: disposing a layer of an adhesion matrix on the substrate; and culturing cells on the adhesion matrix. ### Claim 4 (depends on 3) The method according to claim 3, wherein the adhesion matrix comprises collagen, fibronectin, gelatin, laminin, entactin, or a combination thereof. ### Claim 5 (depends on 2) The method according to claim 2, wherein the cell monolayer comprises cells selected from the group consisting of human hepatocellular carcinoma cells (HepG2), human epithelial cells (HeLa), human airway epithelial cells (HBEC3-KT), Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK), human embryonic kidney cells (HEK 293), mouse brain tissue cells (Neuro-2a), mouse embryotic fibroblast cells (NIH-3T3), mouse macrophage cells (J774), rat pheochromocytoma cells (PC12), Drosophila melanogaster cells (Kc 167), primary human hepatocyltes, primary human keratinocytes, primary mouse hepatocytes, UACC-810 breast cancer cells, MCF-7 breast cancer cells, mesenchymal stem cells (MSCs), and combinations thereof. ### Claim 6 (depends on 1) The method according to claim 1, wherein the generating a layer of the biological material on a substrate comprises generating a monolayer of a biological molecule on the substrate, wherein the biological molecule is selected from the group consisting of a protein, a polypeptide, a peptide, an antibody, an antibody fraction, a nucleic acid, and combinations thereof. ### Claim 7 (depends on 1) The method according to claim 1, wherein the lyoprotectant of the lyo-processing solution is selected from the group consisting of be trehalose, maltose, sucrose, glucose, cellarabinose, ribose, fucose, talose, tagatose, fructose, galactose, isomaltose, nystose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, dextran, maltodextrose, xylobiose, mannitol, heptose, maltodextrin, hydroxyethyl starch, polyglycolic acid, inulin, fructo-oligosaccharides, galacto-oligosaccharides, mannan-oligosaccharides, lactose, cyclodextrins, hydroxyl propyl methyl cellulose, polysucrose and combinations thereof. ### Claim 8 (depends on 7) The method according to claim 7, wherein the lyo-processing solution is a buffer solution comprising: a buffer with a pH of greater than or equal to about 6.5 to less than or equal to about 8.0; greater than or equal to about 500 mM to less than or equal to about 1.9 M trehalose; greater than or equal to about 5 mM to less than or equal to about 100 mM betaine; and greater than or equal to about 5 mM choline to less than or equal to about 200 mM choline. ### Claim 9 (depends on 8) The method according to claim 8, wherein the buffer solution further comprises: a salt selected from the group consisting of K2HPO4, KH2PO4, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, Ca5(PO4)3(OH), and combinations thereof, wherein the salt has a total concentration of greater than or equal to about 100 μM to less than or equal to about 2 mM. ### Claim 10 (depends on 8) The method according to claim 8, wherein the buffer solution further comprises: a sugar polyol selected from the group consisting of glycerol, erythriol, threitol, arbitol, xylitol, ribitol, mannitol, sorbitol, glactitol, fusitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotiitol, maltotetraitol, polyglycitol, polyethylene glycol, and combinations thereof, wherein the sugar polyol has a concentration of greater than or equal to about 10 μM to less than or equal to about 500 mM. ### Claim 11 (depends on 1) The method according to claim 1, wherein the raising the substrate from the solution comprises raising the substrate out of the lyo-processing solution at a rate of greater than or equal to about 5 mm/min to less than or equal to about 500 mm/min. ### Claim 12 (depends on 1) The method according to claim 1, wherein the inert gas is selected from the group consisting of N2, He, Ne, Ar, Kr, Xe, and combinations thereof. ### Claim 13 (depends on 1) The method according to claim 1, wherein the contacting the biological material with an inert gas as it is raised out of the solution comprises contacting the biological material with an insert gas at pressure of greater than or equal to about 5 psi to less than or equal to about 25 psi. ### Claim 14 A method for lyo-processing a biological material, the method comprising: submerging the biological material comprising biological molecules or cells disposed on a substrate in a lyo-processing solution comprising trehalose, betaine, and choline, the lyo-processing solution being substantially free of Na+; raising the biological molecules or cells disposed on the substrate out of the lyo-processing solution; and passing the biological material disposed on the substrate through an environment that is substantially free of oxygen to lyo-stabilize the biological material. ### Claim 15 (depends on 14) The method according to claim 14, further comprising: storing the lyo-stabilized biological material at ambient temperature. ### Claim 16 (depends on 14) The method according to claim 14, wherein the raising the biological material out of the lyo-processing solution comprises raising the biological material disposed on the substrate out of the lyo-processing solution at a rate of greater than or equal to about 5 mm/min to less than or equal to about 500 mm/min and contacting the biological material with an inert gas selected from the group consisting of N2, He, Ne, Ar, Kr, Xe, and combinations thereof as the biological material is raised out of the lyo-processing solution. ### Claim 17 (depends on 16) The method according to claim 16, wherein the biological material comprises biological molecules selected from the group consisting of a protein, a polypeptide, a peptide, an antibody, an antibody fraction, a nucleic acid, and combinations thereof. ### Claim 18 (depends on 16) The method according to claim 16, wherein biological material comprises the of cells. ### Claim 19 A method for lyo-processing a biological material, the method comprising: generating a layer of the biological material on a substrate; submerging the substrate in a lyo-processing solution comprising a lyoprotectant; withdrawing the substrate from the lyo-processing solution at a rate of greater than or equal to about 5 mm/min to less than or equal to about 500 mm/min; and contacting the biological material with an inert gas as the substrate is withdrawn from the lyo-processing solution to generate a lyo-stabilized biological material on the substrate.