# US 11,896,005 B2 · Warming cryopreserved bone

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- Number: US 11,896,005 B2
- Document ID: US11896005B2
- Status: Granted
- Kind: B2
- Application number: 18/155,657
- Filed: 2023-01-17
- Published: 2023-07-27
- Granted: 2024-02-13
- Earliest claimed: 2020-07-18
- Assignees: Ossium Health, Inc.
- Applicants: Ossium Health, Inc.
- Inventors: Erik J. Woods; Brian H. Johnstone
- Web page: https://patentgrep.com/patent/US11896005B2

## CPC

- A01N 1/00: Preservation of bodies of humans or animals, or parts thereof
- A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time
- A01N 1/10: Preservation of living parts
- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A01N 1/165: Pressure processes, e.g. following predefined pressure changes over time
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- C12N 2509/00: Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N 2523/00: Culture process characterised by temperature
- C12N 2537/00: Supports and/or coatings for cell culture characterised by physical or chemical treatment
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to, at least, a vacuum-assisted method for infiltrating cadaver bone with a cryoprotectant and a method for rapidly warming the cryopreserved cadaver bone for bone marrow processing.

## Claims

### Claim 1

A method for warming cadaver bone for providing bone marrow or a derivative thereof, the method comprising:
obtaining a cryopreserved cadaver bone;
dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone; and
transferring the fragments of the cryopreserved bone into a grinding medium having a temperature of from about 35° C. to 45° C. for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20° C.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cryopreserved cadaver bone has a temperature of at least below 0° C. when dividing into fragments.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cadaver bone fragments are warmed to a surface temperature of about 20° C. at a rate of from about 100° C./min to about 500° C./min.

### Claim 4 (depends on 3)

The method of claim 3, wherein the warming rate is greater than about 300° C./min.

### Claim 5 (depends on 3)

The method of claim 3, wherein the warming rate is from about 400° C./min to about 500° C./min.

### Claim 6 (depends on 3)

The method of claim 3, wherein the warming rate prevents ice recrystallization during thawing of the bone fragments.

### Claim 7 (depends on 1)

The method of claim 1, wherein the grinding medium has a temperature of about 37° C. to about 40° C. when the fragments of the cryopreserved bone are transferred to the grinding medium.

### Claim 8 (depends on 1)

The method of claim 1, wherein the grinding medium comprises two or more of a nuclease, human serum albumin (HSA), heparin, an electrolyte medium, and a growth media.

### Claim 9 (depends on 1)

The method of claim 1, further comprising a step of grinding warmed cadaver bone fragments to obtain ground cadaver bone.

### Claim 10 (depends on 9)

The method of claim 9, further comprising a step of filtering the ground cadaver bone, thereby producing a filtered product comprising bone marrow cells and a captured ground bone.

### Claim 11 (depends on 10)

The method of claim 10, further comprising a step of extracting vertebral bone adherent mesenchymal stromal/stem cells (vBA-MSCs) from the captured ground bone.

### Claim 12 (depends on 11)

The method of claim 11, wherein extracting the vBA-MSCs comprises a step of contacting the captured ground bone with a digestion solution.

### Claim 13 (depends on 12)

The method of claim 12, wherein the digestion solution is contacted with the captured ground bone after the step of collecting bone marrow cells.

### Claim 14 (depends on 9)

The method of claim 9, further comprising a step of removing fat from an intermediate product.

### Claim 15 (depends on 9)

The method of claim 9, further comprising a step of collecting bone marrow cells, thereby obtaining extracted bone marrow cells, wherein the extracted bone marrow cells comprise hematopoietic stem cells (HSCs) and mesenchymal stromal/stem cells (MSCs).

### Claim 16 (depends on 15)

The method of claim 15, wherein at least about 50% of the extracted bone marrow cells or a derivative thereof are viable.

### Claim 17 (depends on 16)

The method of claim 16, wherein at least about 70% of the extracted bone marrow cells or a derivative thereof are viable.

### Claim 18 (depends on 15)

The method of claim 15, wherein the number of the extracted bone marrow cells or a derivative thereof extracted from the warmed cadaver bone fragments is at least about 50% of the number extracted from a fresh cadaver bone.

### Claim 19 (depends on 15)

The method of claim 15, wherein the number of viable CD34+ cells extracted from the warmed cadaver bone fragments is at least about 70% of the number extracted from a fresh cadaver bone.

### Claim 20 (depends on 15)

The method of claim 15, wherein the number of cells with proliferative potential obtained from the warmed cadaver bone fragments is at least about 50% of the number obtained from a fresh cadaver bone.

### Claim 21 (depends on 15)

The method of claim 15 wherein at least about 10 million nucleated cells are extracted from the captured ground bone.

### Claim 22

A method comprising:
(a) placing a cadaver bone in a closed container comprising a cryoprotectant solution;
(b) reducing the pressure in the closed container and, optionally, holding the closed container at reduced pressure, to remove at least a portion of the water present in the cadaver bone;
(c) raising the pressure in the closed container and holding the closed container at a raised pressure to allow infiltration of the cryoprotectant solution into the cadaver bone;
(d) removing the cadaver bone from the closed container;
(e) chilling the cadaver bone to a temperature of at least below 0° C., thereby cryopreserving the cadaver bone;
(f) storing the cryopreserved cadaver bone at a temperature of at least below 0° C.;
(g) dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone; and
(h) transferring the fragments of the cryopreserved bone into a grinding medium having a temperature of from about 35° C. to about 45° C. for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20° C.