# US 2020/0399608 A1 · System and method for extraction and cryopreservation of bone marrow

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- Number: US 2020/0399608 A1
- Document ID: US20200399608A1
- Status: Published application
- Kind: A1
- Application number: 17/013,407
- Filed: 2020-09-04
- Published: 2020-12-24
- Earliest claimed: 2019-04-15
- Assignees: Ossium Health, Inc.
- Applicants: Ossium Health, Inc.
- Inventors: Erik John WOODS; Brian H. JOHNSTONE; Dongsheng GU; Aubrey Marie SHERRY; Kelsey Gwen MUSALL; John R. WOODS; James HARDIN; Alan HOOKS
- Web page: https://patentgrep.com/patent/US20200399608A1

## CPC

- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A61B 17/1611: {the two jaw elements being integral with respective elongate shaft elements}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C12M 45/02: {by mechanical forces; Stirring; Trituration; Comminuting (crushing, pulverizing, disintegrating in general B02C)}
- C12M 45/05: {by centrifugation (centrifuges in general B04B)}
- A61B 17/1606: {of forceps type, i.e. having two jaw elements moving relative to each other}
- A61B 2017/00831: {Material properties}
- A61B 2017/00969: {used for transplantation}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

Methods are provided for extracting bone marrow cells from bone obtained from deceased donors, for preparing the bone marrow for cryopreservation and for obtaining desired cells from cryopreserved and fresh bone marrow.

## Claims

### Claim 1

A method for optimizing recovery of stem cells from cadaver bone or cadaver bone fragments, the method comprising:
(a) obtaining cadaver bone or cadaver bone fragments; optionally, processing the cadaver bone into cadaver bone fragments;
(b) submerging the cadaver bone or cadaver bone fragments in a solution comprising bleach, thereby obtaining a bleached bone product; and then
(c) submerging the bleached bone product in a solution comprising hydrogen peroxide, thereby obtaining a treated bone product;
(d) processing the treated bone product to extract bone marrow cells; and
(e) collecting the extracted bone marrow cells, thereby recovering the stem cells contained therein.

### Claim 2 (depends on 1)

The method of claim 1, wherein the bleach is about a 10% bleach solution.

### Claim 3 (depends on 2)

The method of claim 2, wherein the bleach solution yields about 5,000 ppm free chlorine.

### Claim 4 (depends on 3)

The method of claim 3, wherein the cadaver bone or cadaver bone fragments is submerged in bleach for about 10 to about 25 minutes.

### Claim 5 (depends on 1)

The method of claim 1, wherein the hydrogen peroxide is a 3% hydrogen peroxide solution.

### Claim 6 (depends on 1)

The method of claim 1, further comprising a step of transferring the bleached bone product from a container comprising the bleach solution to a container containing the hydrogen peroxide solution.

### Claim 7 (depends on 1)

The method of claim 1, further comprising a step of agitating the bleached bone product within the hydrogen peroxide solution.

### Claim 8 (depends on 1)

The method of claim 1, wherein step (c) of submerging the bleached bone product in a solution comprising hydrogen peroxide comprises:
(i) submerging the bleached bone product in a container containing the hydrogen peroxide solution;
(ii) detecting foam or froth associated with the bleached bone product; and
(iii) repeating steps (b) and/or (c) until no foam or froth is detected.

### Claim 9 (depends on 8)

The method of claim 8, further comprising a step manually removing soft tissue from a bleached bone product that is associated with foam or froth in step (ii).

### Claim 10 (depends on 8)

The method of claim 8, wherein an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.

### Claim 11 (depends on 1)

The method of claim 1, further comprising a step of transferring the treated bone product into a container containing an electrolyte solution.

### Claim 12 (depends on 1)

The method of claim 1, wherein the processing in step (d) comprises grinding the treated bone product to obtain ground cadaver bone, wherein grinding uses a bone grinder in the presence of a grinding medium.

### Claim 13 (depends on 12)

The method of claim 12, wherein the grinding medium comprises two or more of Benzonase®, HSA, heparin, and PLASMA-LYTE A.

### Claim 14 (depends on 12)

The method of claim 12, wherein the grinding medium promotes extraction of bone marrow cells from the ground cadaver bone.

### Claim 15 (depends on 14)

The method of claim 14, wherein the ground cadaver bone is captured on a filter or sieve and a filtrate that passes thru the filter or sieve comprises bone marrow cells which include stem cells.

### Claim 16 (depends on 1)

The method of claim 1, wherein the cadaver bone or the cadaver bone fragments are derived from a vertebral body, an ileum, or a combination thereof.

### Claim 17 (depends on 1)

The method of claim 1, wherein the cadaver bone or cadaver bone fragments are derived from a plurality of cadaver bones from the same donor.

### Claim 18 (depends on 1)

The method of claim 1, wherein the recovered stem cells comprise hematopoietic stem cells.

### Claim 19 (depends on 1)

The method of claim 1, wherein the recovered stem cells comprise mesenchymal stem cells (MSCs).

### Claim 20 (depends on 15)

The method of claim 15, further comprising incubating the captured ground cadaver bone in a digestive solution comprising collagenase and/or neutral protease to extract vertebral bone adherent mesenchymal stromal/stem cells (vBA-MSCs) from the captured ground cadaver bone.