KR20170064136A - Methods and Compositions for Isolating Mononuclear cells from Bone marrow using Hyaluronic acid - Google Patents

Abstract

The present invention provides a composition for separating mononuclear cells from bone marrow through centrifugation comprising hyaluronic acid, a method for separating mononuclear cells from bone marrow by centrifuging bone marrow with hyaluronic acid, and a method for producing bone marrow-derived mesenchymal stem cells do. According to the present invention, since the mononuclear cell-containing layer is formed at the uppermost position after centrifugation, it is possible to easily separate mononuclear cells. Unlike conventional methods, the present invention does not require an additional washing step to remove density gradient substances, and thus enables efficient separation of mononuclear cells. Hyaluronic acid having excellent biocompatibility is used. Therefore, There is an advantage that it can be transplanted to.

Description

(Methods and Compositions for Isolating Mononuclear Cells from Bone marrow using Hyaluronic Acid)

The present invention relates to a composition for separating mononuclear cells from bone marrow through centrifugation comprising hyaluronic acid, a method for separating mononuclear cells from bone marrow by centrifuging bone marrow with hyaluronic acid, and a method for producing bone marrow-derived mesenchymal stem cells .

Stem cell based therapy is the basis of regenerative medicine. Researchers have conducted a study on the bone marrow are considered to be reliable sources of mesenchymal stem cells that can (bone marrow, BM) and mesenchymal stem cells (mesenchymal stem cells, MSCs) obtained from various tissue sources ^1-3. Studies on MSC have been extensively conducted in the research and clinical sectors, and ^4-6 , BM-derived MSCs (BMSCs) can be obtained at high yields through culture.

In order to separate MSCs from the bone marrow, separation of the mononuclear cell layer (MNC layer) formed after centrifugation is required. For the separation of the MNC layer, a density gradient method using the density difference of bone marrow components has been used. Ficoll is a representative density gradient material. Centrifugation of bone marrow with dense gradient material uses density gradients to separate the MNC layer and the RBC layer, because the intrinsic layers of the components are formed due to the difference in density between the components. When the bone marrow is centrifuged together with the dense gradient substance, the MNC layer is formed on the surface layer of the density gradient substance. Since the MNC layer contains hBMSC, the MNC layer can be collected and cultured to obtain bone marrow stem cells.

However, the conventional method as described above has the following problems. First, as shown in FIG. 1, since the MNC layer is located in the middle layer, there is a problem that the cell recovery rate depends on the skill level of the collector that separates the MNC layer. Secondly, since the human body stability of the density gradients is not ensured, the washing process of the separated MNC layer is indispensable for clinical application in order to prevent contamination by the density gradients used. There is a problem in that it is difficult to transplant in the human body immediately after the separation of MNC.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

 Soncini, M., Vertua, E., Gibelli, L., Zorzi, F., Denegri, M., Albertini, A., Wengler, G. S., and Parolini, O. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 1, 296, 2007.  da Silva Meirelles, L., Chagastelles, P. C., and Nardi, N.B. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119, 2204, 2006.  Barry, F. P., and Murphy, J. M. Mesenchymal stem cells: clinical applications and biological characterization. The international journal of biochemistry & cell biology 36, 568, 2004.  Baksh, D., Yao, R., and Tuan, R.S. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem cells 25, 1384, 2007.  Kern, S., Eichler, H., Stoeve, J., Kluter, H., andBieback, K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem cells 24, 1294, 2006.  Posel, C., Moller, K., Frohlich, W., Schulz, I., Boltze, J., and Wagner, D.C. Density gradient centrifugation compromises bone marrow mononuclear cell yield. PLoS One 7, e50293, 2012.

The present inventors have made efforts to develop a novel technique for bone marrow stem cell differentiation, which has the above-mentioned problems. As a result, when the bone marrow was centrifuged together with hyaluronic acid, hyaluronic acid aggregated with the mononuclear cells and located on the uppermost layer, confirming that the mononuclear cell layer could be easily separated (collected), thereby completing the present invention .

Accordingly, it is an object of the present invention to provide a composition for separating mononuclear cells from bone marrow through centrifugation.

It is another object of the present invention to provide a method for separating mononuclear cells from bone marrow.

It is still another object of the present invention to provide a method for producing bone marrow-derived mesenchymal stem cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for separating mononuclear cells from bone marrow through centrifugation, comprising hyaluronic acid.

According to another aspect of the present invention, there is provided a method for separating mononuclear cells from bone marrow through centrifugation, comprising separating mononuclear cells from bone marrow by centrifuging the marrow separated from the in vitro cells with hyaluronic acid . ≪ / RTI >

The present inventors have made efforts to develop a novel technique for bone marrow stem cell differentiation, which has the above-mentioned problems. As a result, when the bone marrow is centrifuged together with hyaluronic acid, hyaluronic acid is coagulated with mononuclear cells and located on the uppermost layer, thereby enabling easy separation (collection) of mononuclear cell layers, thereby completing the present invention .

The present invention is characterized in that the bone marrow is centrifuged together with hyaluronic acid to separate the bone marrow component through centrifugation. Thus, when centrifugation is carried out using hyaluronic acid, it is possible not only to separate mononuclear cells, which have no qualitative difference, from the mononuclear cells separated by using the conventional method using a density gradient substance (phycol) No additional washing steps are required to prevent contamination, which makes it possible to isolate mononuclear cells more efficiently. In addition, since hyaluronic acid having superior biocompatibility is used in the present invention unlike the phycol used in the conventional method, the separated mononuclear cells can be transplanted into the body immediately after the separation.

The "hyaluronic acid", which is a key component of the present invention, is a type of glycosaminoglycan having a structure in which N-acetylglucosamine and glucuronic acid are alternately linked in a chain, and is mainly used as an animal's articular fluid, , Umbilical cord, dermal surface layer, and the like. Hyaluronic acid has excellent biocompatibility and is known to have no interspecies specificity and no tissue or organ specificity.

According to one embodiment of the present invention, the formulation of hyaluronic acid is a hydrogel. Hyaluronic acid can be formulated into a hydrogel through chemical modification or crosslinking in order to improve and complement its physical properties, and a method for producing a hyaluronic acid hydrogel is well known to those of ordinary skill in the art. For example, hydrogel formation through chemical modification of hyaluronic acid and cross-linking can be accomplished through alcohol groups and carboxylic acid groups present in the hyaluronic acid backbone.

According to the present invention, the rate of mononuclear cell recovery after centrifugation can be controlled by controlling the crosslinking rate of hyaluronic acid.

According to an embodiment of the present invention, the crosslinking rate of the hyaluronic acid is 30-60%. According to a more specific embodiment, the crosslinking rate is 40-60%, 45-60% or 45-55%.

According to the present invention, the cell recovery after centrifugation can be controlled by adjusting the volume ratio of bone marrow and hyaluronic acid used for centrifugation.

According to one embodiment of the present invention, the volume ratio of the bone marrow to the hyaluronic acid by centrifugation is 1: 0.15-0.30. According to a more specific embodiment, the volume ratio during the centrifugation is 1: 0.15-0.25.

According to the present invention, the cell recovery rate after centrifugation can be controlled by controlling the centrifugal acceleration.

According to an embodiment of the present invention, the centrifugal acceleration is 300 x g to 1200 x g. According to one more specific embodiment, the centrifugal acceleration is from 400 xg to 1000 x g, 400 x g to 900 x g, 400 x g to 800 x g, 400 x g to 750 x g, 450 x g to 750 x g, 450 x g to 700 x g, or 450 xg to 650 xg.

According to the present invention, the cell recovery rate after centrifugation can be controlled by adjusting the crosslinking rate of hyaluronic acid, the volume ratio of bone marrow and hyaluronic acid used for centrifugation, and the centrifugal acceleration.

According to an embodiment of the present invention, the crosslinking rate of the hyaluronic acid is 30-60%, the volume ratio of the bone marrow and hyaluronic acid is 1: 0.15-0.30, the centrifugal acceleration is 300 xg to 1200 xg. According to a more specific embodiment, the crosslinking ratio of the hyaluronic acid is 45-55%, the volume ratio of the bone marrow and hyaluronic acid is 1: 0.15-0.25, and the centrifugal acceleration is 400 xg to 800 xg to be. By carrying out the method of the present invention under such conditions, mononuclear cells can be isolated from the bone marrow at a high recovery rate (see FIG. 3).

According to one embodiment of the present invention, a method for separating mononuclear cells from the bone marrow of the present invention comprises the steps of:

(a) centrifuging the bone marrow separated in vitro with hyaluronic acid to form separated layers; And

(b) separating the uppermost layer formed in step (a), wherein the separated uppermost layer contains mononuclear cells.

As shown in FIG. 1, hyaluronic acid, which is aggregated with mononuclear cells, is located in the uppermost layer among the layers formed after centrifugation. The uppermost layer can be used as a source of mesenchymal stem cell production. At this time, the uppermost layer may be used for producing stem cells after treating hyaluronidase to degrade hyaluronic acid contained therein.

According to another aspect of the present invention, the present invention provides a method for producing bone marrow-derived mesenchymal stem cells comprising the steps of:

(a) centrifuging the bone marrow separated in vitro with hyaluronic acid to form separated layers;

(b) separating the uppermost layer of the layer formed in step (a), the isolated uppermost layer comprising mononuclear cells; And

(c) separating the mesenchymal stem cells from the mononuclear cell-containing top layer separated in step (b).

The mononuclear cell-containing uppermost layer separated in step (b) may be applied to step (c) by treating hyaluronidase to degrade hyaluronic acid contained therein.

The separation of the mesenchymal stem cells in step (c) of the present invention can be carried out according to the conventional medium used for separating the mesenchymal stem cells from the mononuclear cell population, the culture conditions, the number of subcultures and the separation method can do.

According to an embodiment of the present invention, the mesenchymal stem cell is isolated in step (c) by separating the adherent cells formed by culturing the uppermost layer containing mononuclear cells in a cell culture apparatus. The cell culture apparatus is, for example, a cell culture plate or a cell culture flask.

The features and advantages of the present invention are summarized as follows:

(I) The present invention relates to a composition for separating mononuclear cells from bone marrow through centrifugation comprising hyaluronic acid, a method for separating mononuclear cells from bone marrow by centrifuging bone marrow with hyaluronic acid, and a method for producing bone marrow-derived mesenchymal stem cells ≪ / RTI >

(Ii) According to the present invention, since the mononuclear cell-containing layer is formed at the uppermost position after centrifugation, it is possible to easily separate mononuclear cells.

(Iii) Unlike the conventional method, the present invention does not require an additional washing step to remove density gradient substances, and thus enables efficient separation of mononuclear cells. Because hyaluronic acid having excellent biocompatibility is used, There is an advantage that it can be transplanted into the body immediately after separation.

Figure 1 shows the location of the MNC layer formed after centrifugation using a phycolate or hyaluronic acid hydrogel.
FIG. 2 shows the results of immunophenotyping analysis of MNC isolated using phycol or hyaluronic acid hydrogel.
Figure 3 relates to optimization of BMSC separation conditions using hyaluronic acid hydrogel. (A) 50% cross-linked HA conditions in the primary culture showed higher cell numbers than 0% and 30% cross-linked HA ( ^* p <0.05). (B) WBC showed the same pattern as the cell count result. (C) Cell proliferation in P1 did not differ according to the crosslinking rate of HA (p = 0.836). (AC) data were expressed as mean ± SD (n = 3) based on normalization values of 50% cross-linked HA. (D) 1 primary cell yield of 3 ml HA during culture was higher than significantly from the 1 ml and ^{2 ml HA (** p <0.001} , * p <0.05). (E) WBC showed the same tendency as in the above cell counts. (F) The lowest cell proliferation was seen at 3 ml HA in P1. (DF) data were expressed as mean ± SD (n = 3) based on normalized values of 3 ml HA. (G) 530 xg centrifugation conditions were significantly higher in cell count ( ^** p <0.001) and (H) WBC ( ^* p <0.05) (I) cell proliferation was not significantly different between the groups (p = 0.493). (GI) data were expressed as mean ± SD (n = 3) based on the normalization value of 530 xg centrifugation conditions.
Figure 4 relates to the in vitro characterization of hBMSCs isolated using either Ficoll or 50% cross-linked HA. (A) shows the cell yield. ^* p < 0.05. Data were expressed as means ± SD (n = 3) based on the normalized values of Ficoll. (B) After three consecutive sub-cultures, the phenotype of hBMSC in fibroblast-like morphology was confirmed. Magnification X100, scale bar = 100 μm. Cell proliferation of hBMSCs obtained using (CE) phycol and 50% cross-linked HA. (C) doubling time, (D) CCK-8 analysis and (E) cell cycle analysis did not show any significant difference between the two groups. (FH) Isolated BMSCs showed colony forming ability. (F) Colony formation of two group BMSCs was confirmed by staining with crystal violet after 13 days. (G) The number of colony forming units (CFU) and colony diameter were not significantly different between the two groups. Data are presented as means ± SD.
Figure 5 relates to a comparison of the immunophenotype and differentiation potential of hBMSCs isolated using Ficoll or 50% cross-linked HA. (A) The BMSC surface markers were positive for CD44, CD73, CD105 and CD90 (> 99%) and negative for CD45, STRO-1, CD14 and CD34 (<3%). The data show that there was no difference in cell purity between the two groups. (BD) hBMSCs. (B) The bone differentiation was stained with Alizarin Red S (magnification X 100, scale bar = 100 μm). (C) The total area of mineralized nodules was not different between the two groups. (D) Real-time PCR analysis. The bone-specific genes ALPL and Runx2 were strongly expressed. There was no significant difference between the two groups. (EG) hBMSCs. (E) Adipocyte differentiation was stained with Oil-Red O (magnification X 100, scale bar = 100 μm). (F) The total area of lipid vacuoles was not different between the two groups. (G) Real time PCT analysis. The adipocyte-specific genes LPL and PPAR-γ were strongly expressed. There was no significant difference between the two groups. (HJ) hBMSC. (H) Cartilage differentiation was stained with Alishan Blue (magnification X200, scale bar = 100 μm). The positive staining area of (I) cartilage-specific proteoglycans was significantly higher in 50% cross-linked HA than in Ficoll (* p> 0.05). (J) Real-time PCR analysis. The cartilage-specific genes Aggrecan and SOX9 were strongly expressed. There was no significant difference between the two groups. Data were expressed as means ± SD (n = 3) based on the normalized values of Ficoll.
Figure 6 relates to the in vivo mineralized tissue formation of implanted hBMSCs isolated using Ficoll and HA hydrogels. Histologic analysis of these structures using H & E staining showed that the bivalve and BM-like tissues were present in the BMSCs separated by two methods along the surface of the carrier, but the negative control showed fibrous tissue without any bone or BM-like structure formation No data). The newly formed bones were highly mineralized when viewed under a polarizing microscope and exhibited laminated tissue characteristics. BM-like tissue had hematopoietic-cell-like components such as hematopoietic cells, and there was no significant difference in bone volume and BM formation between the two groups. Magnification X200; Scale bar = 200 占 퐉.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Preparation of human bone marrow aspirates

A human bone marrow aspirate was taken from a vertebrae-containing syringe in 8 patients (2 males, 6 females, mean age 52 years, range 37-59 years).

Separation of hBMNC by density gradient centrifugation using Ficoll

10 ml of hBM aspirate was diluted 1: 1 with DPBS (Dulbecco's phosphate-buffered saline) and laid on density gradient media (Histopaque, Sigma-Aldrich) containing the phycol. After centrifugation (1500 x, 20 min) at room temperature, the buffy coat was collected in a new tube and washed with DPBS (Fig. 1B). Thereafter, centrifugation was performed at 530 x for an additional 5 minutes, the supernatant was washed, and the remaining pellet was resuspended in 2 ml of culture medium. The culture medium consisted of α-MEM (GIBCO) supplemented with 10% FBS (GIBCO), 20 μg / ml gentamycin (GIBCO), 2 mM GlutaMAX-1 100X (Invitrogen) and 100 μM L-ascorbic acid Sigma). Counting the cells in 1.5 ml solution, it was dispensed into T75 5-10 x 10 ⁶ cells / 20 ml with culture medium to the culture flask. The number of cells in the remaining 0.5 ml solution was measured by CBC (complete blood cell count).

Separation of hBMNC by density gradient centrifugation using hyaluronic acid (HA) hydrogel

The carrier used is a stabilized gel type of HA. The raw HA material is Streptococcus equi subsp. zooepidemicus (ATCC39920) was cultured and fermented. The fermented product was purified through precipitation, filtration and freeze-drying to form a sponge form. The purified HA was stabilized by butanediol diglycidyl ether crosslinking. In this experiment, HA with crosslinking ratios of 0%, 30% and 50% was used. HA was then formulated into gel form by sterilization in PBS. HA was purchased from Shin Poong Pharm.

BM aspirate (10 ml) was layered on HA without dilution with DPBS and then centrifuged at room temperature for 5 minutes. This step was repeated to evaluate the effect of various conditions such as HA type, volume and centrifugal acceleration. After separation into several layers, very low density cells bound to HA were collected (Figure 1C). The collected layers were then dissolved in hyaluronidase (MALINDA INJ, BC World Pharm) and washed with 10 ml of culture medium. The remaining pellet was resuspended in 2 ml of culture medium. Counting the cells in 1.5 ml solution, it was dispensed into T75 5-10 x 10 ⁶ cells / 20 ml with culture medium to the culture flask. The number of cells in the remaining 0.5 ml solution was measured by CBC (complete blood cell count).

Primary culture

After 6-7 days, T75 culture flasks were observed at 100X magnification using a phase-contrast inverted microscope (CKX41, Olympus Optical) and adherent cells were morphologically evaluated (non-adherent cells were first Removed when replacing the medium). The culture medium was freshly replaced every 3-4 days, and subculture was performed for 10-15 days when cell number reached about 80-90% confluence. The cells were washed with DPBS to remove the remaining culture medium and the cells were removed by incubation in 0.25% trypsin-EDTA (GIBCO) for 3 minutes. The medium was centrifuged at 530 xg for 5 minutes, the supernatant was washed and the remaining pellet was resuspended in the culture medium. The survival rate and number of adherent cells were confirmed by trypan blue exclusion test using a hemocyte counter. Was then dispensed into the cell 3-6 x 10 ³ cells / cm ² in T75 or T175 flask. The cells were incubated at 37 [deg.] C in a humidified incubator containing 5% CO ₂ .

Cell morphology and proliferation assay

The morphological features of BMNC from P2 to P6 were observed with a phase-contrast reversed-phase microscope (CKX41, Olympus Optical). The number of cells in each passage was counted using a hemocyte counter and the doubling time T _{d was} calculated by the equation T _d = (t ₂ -t ₁ ) × log (2) / log (q ₂ / q ₁ ) ". Where q ₁ and q ₂ are the number of cells at times t ₁ and t ₂ , respectively.

For cell count analysis (CCK-8 kit, CK04, DOJINDO), the cells were plated at 750 cells / well in 96 well plates (Thermo, China) and cultured for 3 or 5 days. 10 μl of CCK-8 solution was incubated for 3 hours, and absorbance was measured at 450 nm using a microplate spectrophotometer (EPOCH, BIOTEK). For cell cycle analysis, 1 × 10 ⁶ cells were collected, washed with DPBS, and fixed in 1 ml of 70% cold ethanol at 4 ° C for 1-2 hours. The pellet was washed twice with DPBS and stained with 50 μg / ml propidium iodide (Sigma) and 10 μg / ml RNase A (Worthington Biochemicals). Cells were observed with FACS instrument (FACSVerse, BD Bioscience) and data were analyzed using the provided software (FACSVerse, BD Bioscience).

Colony-forming unit analysis

Colony forming unit (CFU) analysis was performed to confirm the presence of BMSC in the hydrogel layer of the surface obtained using the isolated MNC layer and HA mediated method obtained using the conventional Phicol method. Cells were plated at 1 × 10 ² cells / dish in a 60 mm culture dish containing the culture medium. After 10-14 days, the plate was fixed with 10% formaldehyde, and then stained with crystal violet (Sigma). The number and size of CFUs were calculated based on observations using rulers.

Comparison of in vitro multipotency of isolated BMSCs using Ficoll and HA hydrogels

Cells were cultured and bone and adipocyte differentiation was induced. The cells of P3 and P5 when a 6-well plate at 1 x 10 ⁵ cells / well dispensed, and cultured until the cells reached sub-confluence (subconfluence). The culture medium used for bone differentiation contained 10% FBS, 20 μg / ml gentamycin, 2 mM GlutaMAX-1 100X, 100 μM L-ascorbic acid, 0.01 μM dexamethasone (Sigma) and 1.8 mM KH ₂ PO ₄ Gt; alpha-MEM &lt; / RTI &gt; was replaced with fresh medium every 3-4 days. For adipocyte differentiation, adipocyte induction and maintenance medium (Lonza) was used every 3-4 days alternately.

For cartilage differentiation, 4 x 10 ⁵ cells (P3 and P5) containing the sample is dispensed to 15 ml conical tube, which was then separated for 5 minutes and centrifuged at 530 xg. After 2-3 days of incubation in the culture medium, the cells were combined to form free-floating cell pellets. The cartilage forming medium (? -MEM) contained 20 μg / ml gentamycin, 1% ITS (BD Bioscience), 0.1 μM dexamethasone, 0.172 mM L-ascorbic acid, 40 μg / ml proline and 10 ng / beta 3 (R & D Systems) and replaced with fresh media every 3-4 days.

After 2-3 weeks of differentiation, osteogenic cells, adipogenic cells and chondrogenic cells were stained with Alizarin Red dye, Oil Red O dye and Alcian blue dye, respectively. The formation of mineralized nodules and the area of lipid vacuoles were analyzed using Adobe Photoshop CS4 software (Adobe Systems).

Quantitative real-time PCR

In order to confirm the differentiation potential of human BMSC (hBMSC), differentiation markers were analyzed by quantitative real time PCR. Primer sets for each differentiation marker were designed using Primer Express software (version 3.0, Applied Biosystems) (Table 1).

After the differentiation period, each cell was obtained with Trizol (Invitrogen) and total cell RNA was prepared. CDNA was synthesized from total RNA isolated using a High Capacity RNA-to-cDNA kit (Applied Biosystems). PCR was performed using a real-time PCR system (StepOne, Applied Biosystems) and a Power SYBR Green PCR Master Kit (Applied Biosystems) under the following conditions: After the initial polymerase activation step at 95 ° C for 10 min, And each cycle included denaturation at 95 ° C for 15 seconds and extension at 60 ° C for 60 seconds. Real-time PCR data were analyzed by the comparative CT method. Relative expression of mRNA was quantified by comparison with internal standard (GAPDH). Each PCR was repeated three times using the same amount of total DNA.

Fluorescence-activated cell sorting (FACS)

To compare the cells isolated by the two methods, the characteristics of cell surface markers of BMSC were confirmed by flow cytometry using FACS. Cells were washed with PBS and incubated with either CD14 (IM0645U), CD45 (A07783), CD34 (IM1870), CD90 (IM1839U) and CD105 (A07414; all Beckman Coulter), CD44 (555479) ) For 1 hour at room temperature. Cells were washed three times and then observed with a flow cytometer (CYTOMICS FC 500, Beckman Coulter).

In-vitro differentiation evaluation

MBCP (macroporous biphasic calcium phosphate) used for ectopic transplantation was 80 mg of HA / TCP ceramic powder (Biomatlante). BMSCs (6 x 10 ⁶ cells / carrier) separated using conventional Ficoll mediations and the HA mediated method of the present invention were pre-cultured for 1.5 hours by mixing with particles at 5% CO ₂ , 37 ° C prior to transplantation. The cell-bearing carrier was subcutaneously transplanted into the back region of immunodeficient mice. Animals were sorted according to the following three experimental conditions: (1) BMSCs isolated using conventional methods were dispensed into MBCP carriers (Ficoll group, n = 4); (2) the removed BMSC was dispensed in MBCP carrier (HA hydrogel group, n = 4), or (3) the untreated MBCP carrier (control group, n = 0). The animals had a recovery period of 8 weeks after surgery and were sacrificed before subsequent analysis.

Block sections were fixed in 10% formalin. Calcination of the sample was performed using 5% EDTA (pH 8.0) and 4% sucrose, rehydrated in a series of ethanol solutions and embedded in paraffin. The most central part of the 6 μm thick was cut at about 50 μm intervals and stained with Masson's trichrome. Histological analysis was performed using a polarizing microscope (BX41, Olympus Optical). Bone-like tissue formation was analyzed using an automated image analysis system (mage-Pro Plus, Media Cybernetics, Silver Spring). The method involves manually showing the border of a newly formed bone on the captured image of the most central portion slide after the software automatically calculates the area within the borderline.

For immunohistochemical analysis, deparaffinized sections were soaked in 3% hydrogen peroxide to inhibit endogenous peroxidase activity, and then primary mouse monoclonal antibodies against human-specific mitochondria diluted 1: 50 in PBS Lt; / RTI &gt; antibody (ab74285, Abcam). The stained sections were observed with an optical microscope and pictures were taken.

Statistical analysis

All in vitro experiments were repeated three times, and the mean and standard deviation of the results were calculated three times. Unpaired t-test was performed to analyze differences between the two groups. Repeated-measures After the ANOVA, Scheffe's comparisons were used for multiple analyzes. p < 0.05 was considered statistically significant.

Experiment result

Evaluation of MNC layer in HA hydrogel

When hBM was separated into three layers using the HA-mediated method, the surface layer contained a HA hydrogel containing several BM components. FACS analysis was performed on the cultured cells derived from the HA layer, and it was confirmed whether or not the estimated stem cells were included in the HA layer. Over 90% of the cells expressed CD44, a typical MSC marker, whereas CD34, a hematopoietic marker, was not expressed. The expression levels of these markers were the same as hBMSCs isolated using conventional phycool mediated methods (Figure 2). The above results show that stem cells were present in the HA layer.

Optimization of BMSC separation condition through HA

Evaluation of cell yield and proliferation activity by crosslinking rate of HA

Three types of HA hydrogels including 0%, 30% and 50% crosslinked HA were used to determine the optimal crosslinking rate to obtain the highest cell yield from BM. The number of white blood cells (WBC) increased with increasing crosslinking ratio, and cell count and CBC were significantly different immediately after separation in three groups. WBC counts were 6.3-fold and 1.6-fold higher ( p <0.05, Figure 3A) in the 50% cross-linked HA group compared to the 0% and 30% cross-linked HA groups and the CBC test values were 11.5-fold and 1.7-fold higher ( p < 0.05, Fig. 3B). The hBMSCs derived from each HA layer were proliferated to compare the biological characteristics of these cells. As a result, the proliferation rate of the isolated BMSCs was not significantly different between the three groups (p = 0.836, Fig. 3C).

Evaluation of cell yield and proliferation activity by separation conditions

The volume of three different HA (1 ml, 2 ml and 3 ml) and centrifugation accelerations (170 xg, 530 xg and 1500 xg) were used to determine optimal separation conditions for 10 ml of hBM. Immediately after separation, cell yield was assessed by CBC and cell count. The number of WBCs in each MNC showed a tendency to increase with the volume of HA: 3 ml HA group was 5 times and 1.5 times higher than 1 ml HA and 2 ml HA group ( p <0.001, Fig. 3D). Also, the pattern of MNC density was not different from that of WBC ( p < 0.001, Fig. 3E). The proliferation potential of cells isolated from each HA layer was assessed by cell counts. As a result, there was a significant difference in P1 between 1 ml and 3 ml of HA (p <0.05, Fig. 3F) When 3 ml of HA was used, it showed low proliferative activity as compared with 1 ml or 2 ml of HA. The above cell proliferation activity evaluation showed that the cell population was heterogeneous when HA of larger volume (i.e., 3 ml) was used.

Comparing the three centrifugal accelerations, the cell yield was 1.8 times higher ( p <0.001, Fig. 3G) in the 530 xg group compared to the 1500 xg group ( p <0.05, Fig. 3H) . On the other hand, after centrifugation, the 3 ml HA group remained a suspension containing a mixture of HA and BM, so the 170 xg group could not be measured (data not shown). Adherent cell proliferation from each MNC layer at P1 did not differ significantly between the 530 xg and 1500 xg groups ( p <0.493, Fig. 3I).

Biological characteristics of BMSC isolated by using phycol and HA hydrogel

Separation efficiency

Cell yields at the time of centrifugation of 10 ml BM at 530 xg with 2 ml of 50% cross-linked HA identified as optimal conditions for the HA mediated method of the invention were compared to conventional phycol mediated methods. The number of MSCs obtained using the HA-mediated method was 60% and 70% of the Ficoll-mediated method in the direct cell count and CBC test, respectively ( p <0.05, Fig. 4A). Although fewer MSCs have been obtained by the HA mediated protocol than conventional methods, HA mediated methods do not require the required wash procedure to avoid phycol contamination (this wash step can result in significant loss of isolated cells ), The HA-mediated method can be considered as a promising method for direct clinical application of isolated MSCs.

Basic features

In the case of the HA-mediated separation technique considered to be a suitable alternative for hBMSC isolation, it is necessary to assess the effect of HA on the biological characteristics of hBMSCs. The hBMSCs isolated using HA were compared in terms of known characteristics of stem cells and BMSCs isolated using conventional phycoal mediated methods. First, cell morphology showed fibroblast-like appearance, and there was no difference between the two methods (Fig. 4B). Proliferative activity as determined by doubling time and CCK-8 assay also showed a similar pattern. Doubling times of both cell types were shortened to P3 and prolonged from P3 to P6 (Fig. 4C). In P5, CCK-8 analysis was carried out after 3 and 5 days of culture, with no significant difference between the two groups (Fig. 4D). To further validate the proliferation, the cell cycle at P5 was assessed using a flow cytometer to determine the percentage of cells at a particular cell cycle stage. The MSC of HA group was G0 / G1 group with 15.01% S period and 9.36% G2 / M group, and there was no significant difference between MSC (17.7% and 8.45%, respectively) of Ficoll group (Fig. 4E).

In addition, colony forming ability was measured at P3 and P5. After 14 days spindle-like cells were observed in both groups (Fig. 4F). There was no difference between the two groups in the CFU (P3, p = 903; P5, p = 0.777; Fig. 4G) and colony diameter (P3, p = 0.757; P5, p = 0.856;

These results show that there is no significant difference in the basic characteristics of stem cells between the two groups of BMSCs.

Immunophenotypic features

Immunophenotyping tests were performed to confirm that the isolated cells contained MSCs. Expression of the human MSC markers (CD44, CD73, CD105 and CD90) was observed in the HA group, but no leukocyte marker (CD45), mononuclear marker (CD14) and hematopoietic cell marker (CD34) were observed. In addition, the overall expression pattern of cell surface markers was similar in both groups (Figure 5A).

Differentiation potential

The ability of BMSC to differentiate into different mesenchymal tissues using Ficoll mediated method and HA mediated method was evaluated in terms of the degree of BMSC directly differentiating into bone, fat and chondrocyte system. Osteogenic differentiation was detected by detecting osteogenic phenotype composed of alizarin-red-S dyed mineralized ECM. As shown in Fig. 5D, the total area of mineralized nodules did not differ significantly between the two groups (Fig. 5B and 5C), such as osteogenic differentiation-related mRNA expression such as ALPL and Runx2. In addition, BMSCs developed into Oil-Red-O ⁺ cells and could undergo adipogenic differentiation. The total area of lipid vacuoles was not different between the two groups (Fig. 5E and 5F). These results were once again confirmed by the expression of adipocyte differentiation-related mRNA such as LPL and PPAR-y (Fig. 5G). Chondrogenic differentiation was confirmed using allyl acid blue staining to detect cartilage-specific proteoglycans (Fig. 5H). Expression of Aggrecan and SOX9 as stained area and cartilage differentiation-related genes was not significantly different between the two groups (FIG. 5I and FIG. 5J).

In vivo mineralized tissue formation of transplanted hBMSCs isolated using Ficoll and HA hydrogels

The Bboy two separate methods in order to verify whether it affects the multipotent (multipotency), calcified tissue, such as the in vivo transplantation of BMSC with HA / TCP carrier and the slopes once Goals (mineralized bone) and BM similar organizations (calcified tissues were observed. At 8 weeks of transplantation, abnormalities such as local inflammation at the surgical site were not observed in the in vivo transplantation model. HA / TCP BMSC were collected for analysis.

Histological analysis of these structures using H & E staining revealed that the bivalve and BM-like tissues were present in the BMSCs separated by two methods along the surface of the carrier (Fig. 6), whereas the negative control group was fibrous Tissues (data not shown). The newly formed bones were highly mineralized when viewed under a polarizing microscope and exhibited laminated tissue characteristics (Fig. 6). BM-like tissue had hematopoietic-cell-like components, and there was no significant difference in bone volume and BM formation between the two groups (Fig. 6).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Methods and Compositions for Isolating Mononuclear cells from          Bone marrow using Hyaluronic acid <130> PN150362 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 1 aggctgtggg caaggtcat 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 2 ggaaggccat gccagtga 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for LPL <400> 3 tggactggct gtcacgggct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for LPL <400> 4 gccagcagca tgggctccaa 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PPAR gamma-2 <400> 5 tcagggctgc cagtttcg 18 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PPAR gamma-2 <400> 6 gcttttggca tactctgtga tctc 24 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ALPL <400> 7 attgaccacg ggcaccat 18 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ALPL <400> 8 ctccaccgcc tcatgca 17 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Runx2 <400> 9 agccctcgga gaggtacca 19 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Runx2 <400> 10 tcatcgttac ccgccatga 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Aggrecan <400> 11 agggcgagtg gaatgatgtt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Aggrecan <400> 12 ggtggctgtg ccctttttac 20 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for SOX9 <400> 13 gacttccgcg acgtggac 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for SOX9 <400> 14 gttgggcggc aggtactg 18 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for collagen 2A1 <400> 15 ggcaatagca ggttcacgta ca 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for collagen 2A1 <400> 16 cgataacagt cttgccccac tt 22

Claims (17)

A composition for separating mononuclear cells from bone marrow through centrifugation, comprising hyaluronic acid.
The composition of claim 1, wherein the formulation of hyaluronic acid is a hydrogel.
The composition of claim 1, wherein the hyaluronic acid is crosslinked and the crosslinking rate is 30-60%.
The composition of claim 1, wherein the volume ratio of the bone marrow to the hyaluronic acid during centrifugation is 1: 0.15-0.30.
The composition of claim 1, wherein the acceleration of the centrifugation is 300 xg to 1200 x g.
2. The method of claim 1, wherein the hyaluronic acid is crosslinked and the crosslinking ratio thereof is 30-60%, the volume ratio of the bone marrow and hyaluronic acid is 1: 0.15-0.30, the acceleration of centrifugation is 300 x g to 1200 x g.
7. The method of claim 6, wherein the hyaluronic acid is crosslinked and the crosslinking ratio thereof is 45-55%. The volume ratio of the bone marrow to hyaluronic acid during centrifugation is 1: 0.15-0.25, and the acceleration of centrifugation is 400 x g to 800 x g.
A method for separating mononuclear cells from bone marrow through centrifugation, characterized by centrifuging the bone marrow separated from the in vitro with hyaluronic acid.
9. The method of claim 8, wherein the method comprises the steps of:
(a) centrifuging the bone marrow separated in vitro with hyaluronic acid to form separated layers; And
(b) separating the uppermost layer formed in step (a), wherein the separated uppermost layer contains mononuclear cells.
9. The method of claim 8, wherein the formulation of hyaluronic acid is a hydrogel.
9. The method of claim 8, wherein the hyaluronic acid is crosslinked and the crosslinking rate is 30-60%.
9. The method of claim 8, wherein the volume ratio of bone marrow to hyaluronic acid in step (a) is 1: 0.15-0.30.
9. The method of claim 8, wherein the centrifugation is performed at an acceleration of 300 x g to 1200 x g.
The method according to claim 8, wherein the hyaluronic acid is crosslinked and the crosslinking ratio thereof is 30-60%, the volume ratio of bone marrow to hyaluronic acid in step (a) is 1: 0.15-0.30, Wherein the acceleration is from 300 x g to 1200 x g.
15. The method according to claim 14, wherein the hyaluronic acid is crosslinked and the crosslinking ratio thereof is 45-55%, the volume ratio of bone marrow to hyaluronic acid in step (a) is 1: 0.15-0.25, Wherein the acceleration is from 400 x g to 800 x g.
A method for producing bone marrow-derived mesenchymal stem cells comprising the steps of:
(a) centrifuging the bone marrow separated in vitro with hyaluronic acid to form separated layers;
(b) separating the uppermost layer of the layer formed in step (a), the isolated uppermost layer comprising mononuclear cells; And
(c) separating the mesenchymal stem cells from the mononuclear cell-containing top layer separated in step (b).
[17] The method according to claim 16, wherein the mesenchymal stem cells are separated from the monocyte-containing uppermost layer by culturing the cells in a cell culture apparatus.

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