WO2008035832A1 - Bone regeneration cell composition and manufacturing method thereof - Google Patents
Bone regeneration cell composition and manufacturing method thereof Download PDFInfo
- Publication number
- WO2008035832A1 WO2008035832A1 PCT/KR2006/003791 KR2006003791W WO2008035832A1 WO 2008035832 A1 WO2008035832 A1 WO 2008035832A1 KR 2006003791 W KR2006003791 W KR 2006003791W WO 2008035832 A1 WO2008035832 A1 WO 2008035832A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- osteogenic capacity
- hyaluronic acid
- bone
- cell
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
Abstract
Provided is a cell composition for promoting bone formation which is capable of achieving local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals; and a method for preparing the same. The method comprises isolating cells having osteogenic capacity from bone marrow; culturing and proliferating the isolated cells in DMEM (Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension having osteogenic capacity; and mixing the resulting cell suspension having osteogenic capacity with a bio-matrix to perform cell-surface treatment for improving the viability of the cells having osteogenic capacity and the mobility/anchorage thereof into bone cavities, thereby preparing a cell therapy product having osteogenic capacity having osteogenic capacity.
Description
[DESCRIPTION] [Invention Title]
BONE REGENERATION CELL COMPOSITION AND MANUFACTURING METHOD THEREOF
[Technical Field]
The present invention relates to a cell composition for promoting bone formation, and a method for preparing the same. More specifically, the present invention relates to a cell composition for promoting bone formation which is capable of achieving local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals. In particular, when a therapeutic cell composition for bone formation according to the present invention is injected via the blood stream, it is possible to achieve uniform delivery of bone- forming cells to target regions in need of bone formation while not causing destruction of a structure of the remaining bone tissue, and thereby it is possible to treat a variety of bone diseases including osteoporosis in need of extensive bone regeneration. Therefore, the present invention accomplishes remarkably improved quality and reliability of the product and thereby is very useful to enhance customer satisfaction.
[Background Art]
As is well known in the art, osteoporosis is the medical term used to describe a condition which undergoes a gradual loss of bone mass and density and as a result, is highly susceptible to bone fracture due to the formation of increased numbers of tiny pores within the bone, as shown in coarse pumice stones or sponges. That is, osteoporosis is a disease of progressive bone loss involving the formation of many tiny holes or pores as compared to normal bone, reduction of bone mass, thinning and weakening of bone microarchitecture, thus causing the bones to become brittle and prone to breaking even with only light impact. Because osteoporosis progresses silently without subjective pain or symptoms, people might not be aware that they have osteoporosis until they accidentally fall or get hit and in turn easily break a bone. Even with light falls, people having osteoporosis may experience wrist fractures, pelvic fractures and vertebral fractures with accompanying severe pain. In particular, pelvic fractures and vertebral fractures are severely painful and require surgical operations, and the patients have to put up with the hardship of being sick in bed even for several months. Even after complete recovery from the surgery, physical impairment may still remain due to surgical sequelae or complications of osteoporosis.
Bones continuously undergo decomposition and replacement processes. That is, remodeling occurs constantly in all bones. During remodeling, old bone is destroyed and absorbed by osteoclasts, and new bone is formed by osteoblasts. If bone absorption exceeds bone regeneration due to imbalanced homeostasis of the bone tissue, this may lead to the occurrence of osteoporosis. It is known that the incidence of osteoporosis has a relationship with combination of various risk factors
such as female gender, a thin and/or small body frame, advanced age, a family medical history of osteoporosis, menopause (including hysterectomy), irregular menstruation (amenorrhoea), neurasthenia, use of adrenocortical hormones or anticonvulsants, hypoandrogenemia in male gender, insufficient exercise, smoking, excessive drinking, Asian and Caucasian nationalities (African and Hispanic- American nationalities are at lower risk), early menopause (before age 45), excessive caffeine and alcohol consumption, and diets low in calcium.
The incidence of osteoporosis is higher in Asian people than American people. Osteoporosis is estimated to affect more than 28,990,000 American people (80 percent of those affected are women). In the United States, 10 million individuals already have osteoporosis. 18 million more have low bone mass, placing them at increased risk for osteoporosis. One in two women and one in eight men over age 50 will have an osteoporosis-related fracture in their lifetime. One in ten African-American women over age 50 has osteoporosis; an additional one in 3 has low bone density that puts them at risk of developing osteoporosis. Osteoporosis is responsible for more than 1.5 million fractures annually, including: 300,000 hip fractures, 700,000 vertebral fractures, 250,000 wrist fractures and 300,000 fractures at other sites.
In the United States, 12,000,000 fracture cases occur each year, 147,000 to 250,000 cases of which are hip fractures and 80% of which is caused by light trauma. By age 80, about 40% of women experience at least one vertebral fracture. A third of women and a sixth of men will experience a hip fracture by the time they are in their late 80s. It is known that 25 to 50% of the hip fracture patients are
unable to walk without the assistance of another person even after hip repair surgery and such fractures are associated with mortality.
Among various therapeutic methods developed to treat the osteoporosis, conventionally-established osteoporosis therapies such as by use of bisphosphonates or selective estrogen receptor modulators (SERMs) primarily focus on the suppression of bone absorption and are known to inhibit a progress of osteoporosis via the prevention of a further loss of bone mass. In addition, by using bone grafting or transplantation of autologous osteoblasts, bone union or bone regeneration can be achieved in fractures of local lesions caused by various factors including osteoporosis or in target regions requiring bone regeneration.
However, the conventional therapeutic methods block a further progress of osteoporosis by preventing bone absorption via inhibition of the osteoclast activity, and therefore suffer from problems failing to substantially facilitate bone regeneration. Further, use of the above-mentioned bone graft technique or autologous osteoblast-based therapy products is disadvantageous in that it is difficult to achieve biological bone regeneration throughout extensive regions.
Even though development of cell therapy products has been driven in order to overcome disadvantages of bone regeneration suffered by the classic bone graft technique, it is difficult to carry out systemic application of adherent cells or therapeutic treatment of adherent cells via the blood stream. This is because it is impossible to carry out cell injection via the blood stream because adherent cells may die if they are not adhered to an adequate substrate.
Reviewing further details of the conventional arts, therapeutic methods using bone allograft, bone autograft or transplantation of autologous osteoblast-
based therapy products for local application have been used when bone defects or osteonecrosis took place in local lesions, or therapeutic methods using bone-absorption inhibitors such as bisphosphonate and the like have been used when bone defects have occurred throughout a broad range of lesions such as osteoporosis. The bone allograft still suffers from the problems such as propagation possibility of contagious diseases, insufficient supply of implant materials, occurrence of undesired immune reaction such as graft rejection, and a difficulty in complete regeneration of the implants into autologous tissues. Meanwhile, the bone autograft solves or alleviates such problems suffered by the bone allograft, but has disadvantages such as a difficulty of securing sufficient donor sites to provide bone for bone transplantation, the morbidity of the donor sites and the like. Cell therapy product utilizing autologous osteoblasts is a therapeutic method which was developed to solve the problems of the conventional bone graft techniques, and is known as a technique which is capable of achieving local bone regeneration by mass proliferation of osteoprogenitor cells isolated from bone marrow, differentiation of the osteoprogenitor cells into osteoblasts and transplantation of the osteoblasts into the target sites in need of bone regeneration.
However, all of the above-mentioned techniques can be applied only for local bone regeneration and simply serve to fill an empty space of the bone, but suffer from disadvantages of difficulty to treat bone defects extensively distributed throughout the body, for example systemic bone defects due to osteoporosis and extensive bone defects due to osteonecrosis. Further, bone-absorption inhibitors used to treat the osteoporosis have no bone regeneration-promoting ability and
thereby suffer from many limitations in treatment of extensive bone damage due to the osteoporosis.
[Disclosure] [Technical Problem]
Therefore, the present invention has been made in view of the problems associated with conventional bone graft techniques as discussed above, and it is a first object of the present invention to provide a method for preparing a cell composition for promoting bone formation by isolating cells having osteogenic capacity from bone marrow, preparing a cell suspension having osteogenic capacity and subjecting the cells to surface treatment, thereby preparing a cell therapy product having osteogenic capacity.
For this purpose, a second object of the present invention is to provide local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals.
A third object of the present invention is to achieve uniform delivery of bone-forming cells to target regions in need of bone formation while not causing destruction of the remaining bone tissue structure, by injection of a bone-forming therapeutic agent via the blood stream. Therefore, it is possible to treat various bone diseases including osteoporosis in need of extensive bone regeneration.
A fourth object of the present invention is to provide a cell composition for promoting bone formation and a method for preparing the same, which are
suited for enhancing customer satisfaction via remarkably improved quality and reliability of the product.
[Technical Solution]
In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a method for preparing a cell composition for promoting bone-formation, comprising:
A) isolating cells having osteogenic capacity from bone marrow; B) culturing and proliferating the isolated cells in DMEM (Dulbecco's
Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension having osteogenic capacity; and C) mixing the resulting cell suspension having osteogenic capacity with a bio-matrix to perform cell-surface treatment for improving the viability of the cells having osteogenic capacity and the mobility/anchorage thereof into bone cavities, thereby preparing a cell therapy product having osteogenic capacity.
In accordance with another aspect of the present invention, there is provided a cell composition for promoting bone-formation, comprising autologous cells having osteogenic capacity and a low-molecular weight hyaluronic acid, wherein the cells are surface-coated with the hyaluronic acid. Herein, the autologous cells having osteogenic capacity are isolated from bone marrow, and are cultured in α-MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid) to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application. Thereafter, the isolated cells are cultured and proliferated in DMEM
(Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to prepare a cell suspension having osteogenic capacity, and the resulting cell suspension is mixed with a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW as a bio-matrix, the hyaluronic acid being added in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing I xIO6 to IxIO7 cells having osteogenic capacity. The low-molecular weight hyaluronic acid is reacted with the cells in a CO2 incubator at a temperature of 36 to 38 °C and a concentration of 4 to 6% CO2 for 1 to 2 hours to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity.
[Description of the Drawings]
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a process flow chart illustrating a method for preparing a cell composition for promoting bone-formation which is applied to the present invention; FIG. 2 is a photograph of a cell composition for promoting bone-formation which is applied to the present invention;
FIG. 3 is an enlarged X-ray photograph of femora and tubercles of rats in Example 1 of the present invention; and
FIG. 4 is a photograph confirming mobility/anchorage of cells having osteogenic capacity injected into blood stream, as in Example 2 of the present invention.
[Best Mode]
Hereinafter, the preferred embodiments of the present invention for accomplishing the above-mentioned objects will be described in more detail with reference to the accompanying drawings. A cell composition for promoting bone-formation and a method for preparing the same, which are applied to the present invention, are constituted as shown in FIGS. 1 and 2.
In connection with description of the present invention hereinafter, if it is considered that description of known functions or constructions related to the present invention may make the subject matter of the present invention unclear, the detailed description thereof will be omitted.
Terms which will be described hereinafter are established taking into consideration functions in the present invention and may vary according to manufacturer's intention or a usual practice in the related art. Therefore, the terms used herein should be defined based on the contents of the specification of the present invention.
First, in further explanation of osteoporosis before entering the detailed description of the constitution of the present invention, the therapeutic method using bone-absorption inhibitors such as bisphosphonates and the like may be applied
when extensive bone defects such as osteoporosis have occurred throughout broad regions. Such a bone allograft technique still suffers from the various problems such as propagation possibility of contagious diseases, insufficient supply of implant materials, occurrence of immune rejection such as graft rejection, and a difficulty in complete regeneration of the implants into autologous tissues. The bone autograft technique solves or alleviates such problems of the bone allograft technique, but also has shortcomings such as a difficulty of securing sufficient donor sites, the morbidity of the donor sites and the like. The autologous osteoblast- based therapy product is a therapeutic method which was developed to solve the problems of such conventional bone graft techniques, and is known as a technique which is capable of achieving local bone regeneration by large-scale growth of osteoprogenitor cells isolated from bone marrow, differentiation of the osteoprogenitor cells into osteoblasts and transplantation of the osteoblasts into the target sites where bone regeneration is sought. However, when a liquid osteoblast suspension is injected into the target site, the autologous osteoblast-based therapy technique may suffer from the problems associated with probability of reduced viability of the osteoblasts, which are adherent cells, and the problems associated with probability of escape of injected osteoblasts from the desired target site for bone regeneration and then propagation thereof to other sites via the blood stream. Therefore, in order to achieve more efficient bone formation, it is necessary to secure the desired viability of the cells and correct delivery of the cells into the target site for treatment. It can be said that the cell therapy product, wherein cells are surface-treated by mixing the cells having osteogenic capacity with a bio- matrix, is a more advanced version of an injection method of a liquid osteoblast
suspension which is based on cell therapy. The conventional injection method of a liquid osteoblast suspension, which involves transplantation of the osteoblasts alone, has suffered from various problems such as decreased viability of the osteoblasts which are adherent cells, and the migration possibility of injected osteoblasts to unwanted regions via the blood stream, rather than toward the desired target sites for bone regeneration. Therefore, in order to achieve more effective osteogenesis, it is desired to secure the desired viability of the cells and correct delivery of the cells into the target treatment sites. In contrast, the three- dimensional cell therapy product for promoting bone formation, which is composed of a combination of cells having osteogenic capacity and a bio-matrix, in accordance with the present invention, due to combination of the cells having osteogenic capacity with the bio-matrix, can ensure a desired viability of the cells and can migrate only into bone defect regions when it is injected into the blood stream and therefore it is possible to effectively treat extensive bone defects such as osteoporosis.
Hereinafter, the constitution of the present invention for the treatment of osteoporosis will be described in more detail.
According to the present invention, provided is a method for preparing a cell composition for promoting bone-formation. The cell composition can be prepared by the following steps.
That is, the present invention provides a step of isolating cells having osteogenic capacity from bone marrow (Step A).
As the cells having osteogenic capacity used in Step A according to the present invention, autologous cells are preferably employed.
Herein, the autologous cells having osteogenic capacity are autologous cells cultured in α-MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid), in order to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application. Further, the present invention provides a step of culturing and proliferating the thus-isolated cells in DMEM (Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension of the cells having osteogenic capacity (Step B).
Thereafter, the present invention provides a step of mixing the resulting cell suspension having osteogenic capacity with a bio-matrix to perform cell- surface treatment for improving the viability of the cells having osteogenic capacity and the mobility/anchorage thereof into bone cavities, thereby preparing a cell therapy product having osteogenic capacity (Step C).
The present invention is also characterized by using a low-molecular weight hyaluronic acid of 1 ,000 to 1 ,000,000 MW as the bio-matrix in Step C.
Then, the present invention provides steps of adding the low-molecular weight hyaluronic acid in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing l*106 to IxIO7 cells having osteogenic capacity; reacting the low-molecular weight hyaluronic acid with the cells in a CO2 incubator at a concentration of 4 to 6% CO2 and a temperature of 36 to 38 °C for 1 to 2 hours, in order to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity; and removing the hyaluronic acid not attached to the cell surface to thereby prepare a cell composition for promoting bone-formation.
Further, the present invention provides a cell composition for promoting bone-formation, comprising autologous cells having osteogenic capacity and a low-molecular weight hyaluronic acid wherein the cells are surface-coated with the hyaluronic acid, through the above-mentioned steps. In particular, the present invention is characterized by the following: the autologous cells having osteogenic capacity are isolated from bone marrow, and are cultured in α-MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid) in order to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application. Thereafter, the isolated cells are cultured and proliferated in DMEM (Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension having osteogenic capacity, and the resulting cell suspension having osteogenic capacity is mixed with a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW as a bio-matrix, the hyaluronic acid being added in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing IxIO6 to IxIO7 cells having osteogenic capacity. The low-molecular weight hyaluronic acid is reacted with the cells in a CO2 incubator at a concentration of 4 to 6% CO2 and a temperature of 36 to 38 °C for 1 to 2 hours, in order to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity. According to the present invention, the hyaluronic acid is attached to the surface of the autologous cells, such that the cell composition for promoting bone- formation can be injected into the blood stream via an artery or vein.
Hereinafter, embodiments and effects of a cell composition for promoting bone-formation according to the present invention and a method of preparing the same, as constituted above, will be illustrated.
First, according to the present invention, attachment of a low-molecular weight hyaluronic acid having 1,000 to 1,000,000 MW to CD44 present on the surface of the cells having osteogenic capacity leads to an increased survival time of cells because the cells having osteogenic capacity which are adherent cells can survive only in the presence of an adequate support material, and also leads to an improved mobility of the cells into the bone cavities when the low-molecular weight hyaluronic acid are attached to the cells having osteogenic capacity. Whereas, attachment of a high-molecular weight hyaluronic acid to the cells results in an increased volume of the cells and a decreased mobility of the cells in the blood stream due to aggregation thereof. In addition, if the low-molecular weight hyaluronic acid is added in a ratio of less than 0.01% per 10 to 20 mL of the cell suspension, the amount of the low-molecular weight hyaluronic acid attached to the cells having osteogenic capacity and the number of cells bound to the hyaluronic acid are decreased, thereby resulting in a decrease of an therapeutic efficiency. Therefore, the low-molecular weight hyaluronic acid was added in a ratio of 0.01% per 10 to 20 mL of the cell suspension. In addition, upon binding of the low-molecular weight hyaluronic acid to the cells having osteogenic capacity, the cells should be continuously viable. In order to ensure that the cells do not stay and adhere to the reaction vessel after attachment of the low-molecular weight hyaluronic acid to the cells, the cells and hyaluronic acid are reacted in a CO2 incubator furnished with favorable growth
conditions at a concentration of 5% CO2 and a temperature of 37 °C . In addition, the cells are reacted for an optimal reaction time of 1 to 2 hours, taking into consideration the time necessary for attachment, anchorage and growth of the suspended cells having osteogenic capacity onto a supporting material. In addition, because the low-molecular weight hyaluronic acid unbound to the cells having osteogenic capacity probably has adverse side effects on the viscosity of the therapeutic agent and the cell viability, due to aggregation of hyaluronic acid which may occur during the production process of the therapeutic agent, the unbound hyaluronic acid is precipitated by centrifugation and is removed from the hyaluronic acid-bound cells by washing the cells twice or more. In order to enhance the cell viability until surgical application, the thus-washed low- molecular weight hyaluronic acid-bound cells having osteogenic capacity are suspended in an injection solution of DMEM (Dulbecco's Modified Eagle's Medium) containing 12.5 to 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid) serving as a pH buffer.
1. Collection of bone marrow
Collection of iliac crest from animals: individual difference, 20 to 50 mL of bone marrow was collected from the iliac crest in case of human. ( 1 ) Composition of solution used for collection of bone marrow
- Low-glucose DMEM, 450 mL
- FBS, 50 mL
- L-glutamine, 5 mL
- Gentamicin (100 μg/mL), 1 mL
- Heparin (10 IU/niL) 500 μi → HBSS 10 niL + 100,000 unit heparin
- HEPES, 25 mM
(2) The collected bone marrow is mixed with the solution used upon harvesting the iliac crest, stored in a refrigerator and transferred.
2. Isolation of nucleated cells (1) Cell culture in bone marrow
- Hank's Balanced Salt Solution (HBSS): containing 100 μg/mL of gentamicin and 2.5 βglmL of fungizone added to a final concentration.
- 10% low-glucose DMEM (with gentamicin and fungizone)
- Antibiotic concentration: final concentration of 50 μg/mL gentamicin and 2.5 /zg/mL fungizone
1) A transfer bottle is washed with 5 mL of HBSS and transferred to a 50 mL tube.
2) Centrifugation is carried out at 1200 rpm for 5 min.
3) The centrifugation product is washed.
4) A 50 mL fresh tube is equipped with a sieve. 5) 10 mL of HBSS is added to the tube which is then closed with a stopper and is vigorously tapped 50 times.
6) Supernatant is decanted and sieved to collect.
7) 10 mL of HBSS is added again to the tube which is then closed with a stopper and is vigorously tapped 50 times.
8) Supernatant is decanted and sieved to collect.
9) The thus-pooled supernatant (30 niL + 10 mL soup of cut bones) is centrifuged at 1200 rpm for 5 min.
10) The resulting supernatant is discarded, and the remainder is suspended in 10 mL of 10% low-glucose DMEM (containing an antibiotic), is divided into two aliquots and is seeded into each T-25 flask, followed by incubation at a temperature of 37 "C and a concentration of 5% CO2.
(2) Cell culture from bones in bone marrow - 0.2% collagenase: Type II collagenase is dissolved to a concentration of
0.2% in HBSS and is then filtered through a 0.2 μm syringe filter.
- 10% FBS low-glucose DMEM (with a final concentration of 100 βg/mL gentamicin): for neutralization and washing.
- 10% FBS low-glucose DMEM (with gentamicin and fungizone), an antibiotic concentration: a final concentration of 50 βg/mL gentamicin and 2.5
/zg/mL fungizone.
1) 10 mL of HBSS is added to the bones remained after decantation of the supernatant in Step 8 of Method (1), the mixture is transferred to a dish, muscles and foreign materials adhered to the bones are removed and the bones are cut. 2) The bones are only transferred to a 50 mL tube to which 3 mL of 0.2% collagenase solution is then added and the tube is allowed to stand at 37°C over night.
3) Foreign materials except for the bones are discarded, and the remaining soup is returned to Step 8 of Method 1.
4) 10 mL of 10% low-glucose DMEM is added to neutralize the soup.
5) Supernatant is decanted and sieved to collect.
6) 10 mL of 10% low-glucose DMEM is added to the soup which is then shaken, and supernatant is decanted and sieved to collect. 7) 10 mL of 10% low-glucose DMEM is added again to the tube which is then shaken, and supernatant is decanted and sieved to collect.
8) The thus-pooled supernatant is centrifuged at 1200 rpm for 5 min.
9) The resulting supernatant is discarded, and the remainder is suspended in 10 mL of 10% low-glucose DMEM (containing an antibiotic), is divided into two aliquots and is seeded into each T-25 flask, followed by incubation at a temperature of 37 °C and a concentration of 5% CO2.
3. Details of Culture method
Isolated nucleated cells are cultured and proliferated in 10% FBS Io w- glucose DMEM. Upon performing cell subculture taking a period of 3 to 4 weeks, a predetermined number of cells are stored.
1) Subculture
1. Isolated nucleated cells are washed with HBSS three times. 2. The cells are treated with 2 or 4 mL of trypsin and incubated for 3 to 5 min.
3. The cell cultures are treated with 1 or 2 mL of FBS, and are neutralized with shaking.
4. The cells are filtered (through a cell strainer), transferred to a 15 mL tube, washed with 10 mL of a medium, and centrifuged at 1,200 rpm for 5 min.
5. The cells are subjected to suction, tapping and suspension, then collected in a tube, and washed with 5 mL of a medium. 6. The cells are seeded in a flask.
7. The remaining cells are stocked.
2) Freezing protocol
1. A freezing medium (FBS:DMSO = 9:1), which was previously freeze- stored, is warmed and stored in a refrigerator at a temperature of 4 °C .
2. The cells are observed under a microscope, QC samples are collected on a clean bench.
3. The culture medium is removed, washed with a given amount of an HBSS solution. This procedure is repeated three times. 4. Each vessel is treated with trypsin and is incubated for 5 min.
5. The cell cultures are neutralized with FBS and shaken. Then, the culture flask is washed with a medium, and the cell cultures are transferred to a 15 mL tube and centrifuged for 5 min.
6. Supernatant is discarded and the cells are suspended in 10 mL of a medium. Cell count is carried out using a Coulter counter.
7. After centrifugation for 5 min, the cells are mixed with a freezing medium and each 1.8 mL aliquot having a cell density of 2X106 to 1.5X107 cells is stored in vials.
8. Upon performing transplantation, the cells may be stored at a temperature of 4°C for up to 2 hours.
9. The cells are freeze-stored in a double-seal container at -70 °C overnight (up to 72 hours). 10. On the next day, the cells are stored in a liquid nitrogen tank at -196 °C for a long period of time.
4. Detailed characteristics of cell culture for a predetermined period In order to enhance an efficiency of the therapeutic agent by improving functions of the autologous cells having osteogenic capacity as osteoprogenitor cells, the cells are cultured by replacement of a culture medium with 10% FBS α- MEM containing 50 mg/L of vitamin C (ascorbic acid), 7 to 10 days prior to surgical transplantation.
5. Reaction of cells with low-molecular weight hyaluronic acid
Cell collection
1) Cells cultured in each T-150 flask (n = 5) are treated with trypsin and collected (filtration through a cell strainer).
2) The collected cells are counted and 1 x 107 cells are taken. 1. 50 μi of DMSO is aliquoted to a tube containing 50 βg of CM-DiI
(3H-Indolium, 5-[[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(l,3-dihydro-3,3- dimethyl- 1 -octadecyl-2H-indol-2-ylidene)- 1 -propenyl] -3,3 -dimethyl- 1 -octadecyl chloride).
2. 25 fd of CM-DiI is added to 2 mL of HBSS, and the cells are made ready for fluorescent labeling.
3. The collected cells are treated with CM-DiI and are aliquoted into Petri dishes having a diameter of 10 cm (25 μl of DMSO/2 mL of HBSS). 4. The cells are incubated at 37 °C for 5 min.
5. The cells are incubated at 40C for 15 min. 6. 1 mL of FBS is added to terminate fluorescent labeling. ^ Cell-containing Petri dishes are wrapped with aluminum foil to block light, and the following steps are carried out. 7. The cells are centrifuged at 1200 rpm for 5 min, and washed with HBSS.
8. The cells are centrifuged again at 1200 rpm for 5 min, and washed with HBSS.
9. The cells are suspended in 20 mL of a medium (10% FBS low-glucose DMEM) to which 2 mL of HA was added. 10. The cells are incubated at 370C for about 90 min.
11. 30 mL of HBSS is added and the cells are collected.
12. Centrifugation (1200 rpm, 5 min) is carried out to remove a supernatant, thereby leaving coated cells.
13. DMEM containing 20 mL of 12.5 mM HEPES is added to re-suspend the cells.
14. After centrifugation, the cells are washed twice with low-glucose DMEM containing 12.5 mM HEPES.
15. The resulting cell pellets are suspended in low-glucose DMEM containing 400 μl of 12.5 mM HEPES.
16. The cell suspension is wrapped with foil (to block fluorescent labeling) and transported while maintaining it at a temperature of 4°C .
6. Reasons why reaction and incubation are conducted in a CO2 incubator at 37°C for l to 2 hours
When the low-molecular weight hyaluronic acid is bound to the cells having osteogenic capacity, the cells should be continuously viable. In order to ensure that the cells do not stay and adhere to the reaction vessel after attachment of the low-molecular weight hyaluronic acid to the cells, the cells and hyaluronic acid are reacted in a CO2 incubator furnished with favorable growth conditions at a concentration of 5% CO2 and a temperature of 37°C . In addition, the cells are reacted for an optimal reaction time of 1 to 2 hours, taking into consideration the time for which the suspended cells having osteogenic capacity adhere to a supporting material and stably stay to grow therein.
7. Reasons of washing and hyaluronic acid removal
Since the low-molecular weight hyaluronic acid unbound to the cells having osteogenic capacity may have adverse side effects on the viscosity of the therapeutic agent and the cell viability, due to aggregation of the hyaluronic acid which may occur during the production process of the therapeutic agent, the unbound hyaluronic acid is precipitated by centrifugation and the hyaluronic acid- bound cells are washed twice or more to remove the unbound hyaluronic acid.
[Mode for Invention]
EXAMPLES
Now, the present invention will be described in more detail with reference to the following examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.
Example 1 : Application of cells having osteogenic capacity to rat
Bone marrow was collected from iliac crest of rats as an animal model and was subjected to enzyme treatment, and nucleated cells were isolated. An osteogenic inducer was treated to differentiate the nucleated cells into the cells having osteogenic capacity and the cells were then mass-cultured. As a hyaluronic acid for surface treatment of the cells, a low-molecular weight hyaluronic acid
(LMW-HA) of less than 1,000,000 MW was used. The hyaluronic acid was added and uniformly distributed in a vessel in which the cells having osteogenic capacity and a culture medium were mixed. The resulting mixture was reacted in a CO2 incubator at 37 °C for 90 min to effect surface treatment of the cells. The LMW-
HA-surface treated cells having osteogenic capacity were injected into an artery and vein of rats with ovariectomy (OVX)-induced osteoporosis.
Results
By injecting the LMW-HA-surface treated cells having osteogenic capacity were into an artery and vein of rats having OVX-induced osteoporosis as a typical
example of extensive bone loss, therapeutic effects of the cells on osteoporosis were confirmed as shown in FIG. 3.
As can be confirmed from FIG. 3, upon comparing with a femur of the rat (FIG. 3B) having OVX-induced osteoporosis, rats (FIGS. 3C, 3D and 3E), in which the cells having osteogenic capacity were injected into the vein and artery, have exhibited a low degree of bone loss. In addition, as compared to the rat (FIG. 3C) to which non-surface treated cells were injected, the rats (FIGS. 3D and 3E) to which the LMW-HA-surface treated cells were injected exhibited healing of osteoporosis. In FIG. 3, A represents a normal rat; B represents a rat with OVX-induced osteoporosis; C represents a rat with OVX-induced osteoporosis in which the cells having osteogenic capacity were injected into the vein; D represents a rat with OVX- induced osteoporosis in which LMW-HA-surface treated cells having osteogenic capacity were injected into the vein; and E represents a rat with OVX-induced osteoporosis in which LMW-HA-surface treated cells having osteogenic capacity were injected into the artery, respectively.
Example 2: Application of human-derived cells into NOD/SCID mice (non-obese diabetic severe combined immuno-deficiencv mouse) A low-molecular weight hyaluronic acid (LMW-HA) was added and uniformly distributed in a reaction vessel in which human cells having osteogenic capacity and a culture medium were mixed. The resulting mixture was reacted in a CO2 incubator at a temperature of 37 °C and a concentration of 5% CO2 for 120 min to effect surface treatment of the cells. Fluorescence-labeled, human-derived cells
having osteogenic capacity were injected into immunodeficient NOD/SCID mice via the blood stream, and the cell mobility of the human-derived cells and the distribution and anchorage of the cells within the bone were confirmed as shown in results of FIG. 4. In order to confirm the mobility, distribution and anchorage of the fluorescence-labeled, LMW-HA-surface treated cells having osteogenic capacity, migrated to the femur of the mice via the blood vessels, the fluorescence-labeled cells (C) were observed by enlarging cross sections (A and B) of the mouse femur under a fluorescence microscope, thus confirming that the fluorescence-labeled, LMW-HA-surface treated cells having osteogenic capacity, which were injected to the blood stream, have migrated and settled into the bone.
[Industrial Applicability]
As apparent from the above description, the present invention enables achievement of bone regeneration throughout extensive regions by delivery of bone-forming cells via the blood stream, using a method for preparing a cell therapy product by isolating cells having osteogenic capacity from bone marrow, preparing a cell suspension having osteogenic capacity and subjecting the cells to surface treatment, thereby preparing a cell therapy product having osteogenic capacity.
In particular, the present invention can accomplish local or systemic bone regeneration by subjecting cells having osteogenic capacity to surface treatment
using a hyaluronic acid and injecting the thus-treated cells into a circulation system of target animals.
In addition, the present invention can achieve uniform delivery of bone- forming cells to target regions in need of bone formation while not causing destruction of the remaining bone tissue structure, by injection of a bone-forming therapeutic agent via the blood stream. Therefore, it is possible to treat various bone diseases including osteoporosis in need of extensive bone regeneration.
Therefore, the present invention accomplishes remarkably improved quality and reliability of the product and thereby is very useful to enhance customer satisfaction.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Claims
[Claim 1] A method for preparing a cell composition for promoting bone- formation, comprising: A) isolating cells having osteogenic capacity from bone marrow;
B) culturing and proliferating the isolated cells in DMEM (Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to thereby prepare a cell suspension having osteogenic capacity; and
C) mixing the resulting cell suspension having osteogenic capacity with a bio-matrix to perform cell-surface treatment for improving the viability of the cells having osteogenic capacity and the mobility/anchorage thereof into bone cavities, thereby preparing a cell therapy product having osteogenic capacity.
[Claim 2] The method according to claim 1, wherein the cells having osteogenic capacity in Step A are autologous cells.
[Claim 3] The method according to claim 2, wherein the autologous cells having osteogenic capacity are the autologous cells cultured in α-MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid), in order to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application.
[Claim 4] The method according to claim 1 , wherein the bio-matrix in Step
C is a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW.
[Claim 5] The method according to claim 4, wherein Step C includes: adding the low-molecular weight hyaluronic acid in a ratio of 0.01 to 0.03% per 10 to 20 mL of the suspension containing I xIO6 to IxIO7 cells having osteogenic capacity; reacting the low-molecular weight hyaluronic acid with the cells in a CO2 incubator at a concentration of 4 to 6% CO2 and a temperature of 36 to 38 "C for 1 to 2 hours, in order to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity; and removing the hyaluronic acid not attached to the cell surface.
[Claim 6] A cell composition for promoting bone-formation, comprising autologous cells having osteogenic capacity and a low-molecular weight hyaluronic acid wherein the cells are surface-coated with the hyaluronic acid.
[Claim 7] The composition according to claim 6, wherein the autologous cells having osteogenic capacity are isolated from bone marrow, and are cultured in α-MEM containing 40 to 60 mg/L of vitamin C (ascorbic acid) to improve functions of osteoprogenitor cells, 7 to 10 days prior to a surgical application; the isolated cells are cultured and proliferated in DMEM (Dulbecco's Modified Eagle's Medium) or α-MEM (Minimum Essential Medium, Alpha Modification) to prepare a cell suspension having osteogenic capacity; the resulting cell suspension having osteogenic capacity is mixed with a low-molecular weight hyaluronic acid of 1,000 to 1,000,000 MW as a bio-matrix, the hyaluronic acid being added in a ratio of 0.01 to 0.03% per 10 to 20 mL of the cell suspension containing I xIO6 to IxIO7 cells having osteogenic capacity; and the low- molecular weight hyaluronic acid is reacted with the cells in a CO2 incubator at a temperature of 36 to 38°C and a concentration of 4 to 6% CO2 for 1 to 2 hours to achieve binding between the hyaluronic acid and CD44 (cluster designation 44) present on the surface of the cells having osteogenic capacity.
[Claim 8] The composition according to claim 7, wherein the hyaluronic acid is attached to the surface of the autologous cells, such that the cells can be injected into blood stream via an artery or vein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020060091325A KR100834718B1 (en) | 2006-09-20 | 2006-09-20 | bone recovery cell composition and manufacture method thereof |
| KR10-2006-0091325 | 2006-09-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008035832A1 true WO2008035832A1 (en) | 2008-03-27 |
Family
ID=39200645
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2006/003791 WO2008035832A1 (en) | 2006-09-20 | 2006-09-25 | Bone regeneration cell composition and manufacturing method thereof |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR100834718B1 (en) |
| WO (1) | WO2008035832A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016042572A1 (en) * | 2014-09-19 | 2016-03-24 | Jaswantrai Doshi Vishal | A method of inducing bone formation by ex-vivo osteoblast culturing for implantation |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100007180A (en) * | 2008-07-11 | 2010-01-22 | 세원셀론텍(주) | Method manufacture of bone recovery collagen gel composition |
| KR101037002B1 (en) * | 2008-09-30 | 2011-05-25 | 세원셀론텍(주) | The Specific Culture Media for Chondrocyte Cells and Methods of Uses Thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5902741A (en) * | 1986-04-18 | 1999-05-11 | Advanced Tissue Sciences, Inc. | Three-dimensional cartilage cultures |
| WO2003015802A1 (en) * | 2001-08-14 | 2003-02-27 | Medipost, Co., Ltd. | Composition for treatment of articular cartilage damage |
| EP1338291A2 (en) * | 2002-02-21 | 2003-08-27 | Ed. Geistlich Söhne Ag Für Chemische Industrie | Resorbable extracellular matrix for reconstruction of bone |
| KR20030081821A (en) * | 2002-04-13 | 2003-10-22 | 주식회사 셀론텍 | Artificial culture of osteoblast |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100443413B1 (en) * | 2001-07-30 | 2004-08-09 | 김민지 | A producting method of alcohol lamp |
-
2006
- 2006-09-20 KR KR1020060091325A patent/KR100834718B1/en active IP Right Grant
- 2006-09-25 WO PCT/KR2006/003791 patent/WO2008035832A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5902741A (en) * | 1986-04-18 | 1999-05-11 | Advanced Tissue Sciences, Inc. | Three-dimensional cartilage cultures |
| WO2003015802A1 (en) * | 2001-08-14 | 2003-02-27 | Medipost, Co., Ltd. | Composition for treatment of articular cartilage damage |
| EP1338291A2 (en) * | 2002-02-21 | 2003-08-27 | Ed. Geistlich Söhne Ag Für Chemische Industrie | Resorbable extracellular matrix for reconstruction of bone |
| KR20030081821A (en) * | 2002-04-13 | 2003-10-22 | 주식회사 셀론텍 | Artificial culture of osteoblast |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016042572A1 (en) * | 2014-09-19 | 2016-03-24 | Jaswantrai Doshi Vishal | A method of inducing bone formation by ex-vivo osteoblast culturing for implantation |
| US10577587B2 (en) | 2014-09-19 | 2020-03-03 | Regrow Biosciences Private Limited | Method of inducing bone formation by ex-vivo osteoblast culturing for implantation |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20080026378A (en) | 2008-03-25 |
| KR100834718B1 (en) | 2008-06-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yin et al. | Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles | |
| Arinzeh et al. | Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect | |
| JP6130588B2 (en) | Mesenchymal stem cell-hydrogel-biodegradable or mesenchymal stem cell-hydrogel-non-degradable support composition for skin regeneration or wound healing | |
| JP3808900B2 (en) | Biological material composed of a three-dimensional biocompatible and biodegradable matrix comprising an effective culture of bone marrow stem cells partially or fully differentiated into connective tissue cells and a hyaluronic acid derivative | |
| US11801331B2 (en) | Composition for cartilage regeneration and preparing thereof | |
| JP2001502905A (en) | Formation of cartilage tissue using cells isolated from Wharton's jelly | |
| US7416889B2 (en) | Methods and compositions for repairing cartilage | |
| JP2003517259A (en) | Method for isolating bone and precursor chondrocytes | |
| CZ20032082A3 (en) | Enhanced in vitro cultivation process of mammalian cells for autologous implantation and transplantation methods | |
| Strong et al. | Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells | |
| JP5554002B2 (en) | Method for producing cartilage tissue regeneration sheet | |
| US20160046903A1 (en) | Method for preparing chondrocytes | |
| US20160067377A1 (en) | Stem Cell Seeded Natural Substrates and Methods Relating Thereto | |
| WO2013146992A1 (en) | Method for producing pluripotent stem cells derived from dental pulp | |
| JP5890222B2 (en) | Cultured cartilage tissue material | |
| KR20170108325A (en) | Composition and method for differentiation adult stem cells into chondrocytes | |
| JP7273419B2 (en) | Biomaterial containing adipose-derived stem cells and method for producing the same | |
| JP7273418B2 (en) | Biomaterial for scaffold-free structure containing adipose-derived stem cells and method for producing the same | |
| WO2008035832A1 (en) | Bone regeneration cell composition and manufacturing method thereof | |
| JP7350344B2 (en) | Method for producing chondrocyte pellets from human induced pluripotent stem cells and its uses | |
| Yoshikawa et al. | Bone and soft tissue regeneration by bone marrow mesenchymal cells | |
| Bohlouli et al. | Tissue buccal fat pad–stromal vascular fraction as a safe source in maxillofacial bone regeneration: A clinical pilot study | |
| KR20110032433A (en) | The method of manufacturing the transplantable spheroids of mixed cellular complexes for cell transplantation and the usage of the same | |
| EP3795677A1 (en) | Composition for promoting stem cell differentiation, comprising progenitor cell culture solution and multilayer graphene film, and use thereof | |
| WO2016088373A1 (en) | Cultured cell sheet for transplantation use, method for producing cultured cell sheet for transplantation use, and method for producing bone tissue for transplantation use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 06798874 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 06798874 Country of ref document: EP Kind code of ref document: A1 |