KR101512383B1 - Process of fractionating umbilical cord blood wastes and cell culture media composition comprising the fraction obtained therefrom - Google Patents

Abstract

The present invention relates to a method for producing a hematopoietic stem cell, which comprises centrifuging a umbilical cord blood mixture separated from cord blood (umbilical cord blood) by centrifugation, filtering the hemolymph, filtering the hemolymph by ultrafiltration and nano- And a method for fractionating the useful component of the cell culture medium according to the size of the molecular weight and using various kinds of fractions separated according to the molecular weight size obtained therefrom as a composition for nutrient supplementation of a cell culture medium derived from human and animal, In addition, the effect of proliferating cells derived from an animal can be obtained, and in particular, the use of fetal bovine serum can be reduced or eliminated in a cell culture medium, thereby reducing the risk of contamination originating from an animal.

Anticoagulant, Plasma Enhancer, Cord blood Blood mixture, Cord blood, Molecular weight, Fraction

Description

TECHNICAL FIELD [0001] The present invention relates to a method for fractionating a cord blood blood mixture, and a fraction obtained therefrom. BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a composition for cell culture medium supplement,

The present invention relates to a method for separating a mononuclear cell layer (buffy coat) from umbilical cord blood (umbilical cord blood) collected from a mother with the consent of a mother and keeping it frozen for a long period of time. There is provided a method for fractionating a useful component having a physiological activity by size of molecular weight by a stepwise filtration process including centrifugation, microbial filtration, ultrafiltration, nanofiltration, and bactericidal filtration, and various kinds of fractions obtained therefrom, And to be used as a nutritional supplement composition of cell culture medium.

When the mother gives birth to the newborn, the placenta and the umbilical cord remain. The umbilical cord and placenta are called umbilical cord blood or umbilical cord blood. In the case of umbilical cord blood, it is collected from the umbilical cord at birth, so it can be used at any time when needed. The biggest weakness is that the amount of blood that can be collected is very small. The umbilical cord blood contains hematopoietic stem cells and mesenchymal stem cells, and the storage industry, which separates them and stores them frozen for a certain period of time and provides them when necessary, has been commercialized.

Since the finding of many hematopoietic stem cells in the umbilical cord blood in the early 1980s, the value of the hematopoietic stem cell derived hematopoietic stem cells derived from the umbilical cord blood was used, The value of which has risen rapidly. Currently, Korea's umbilical cord blood storage banking business is the stage of maturing the market formation. It is a form to save the cord blood of the family by giving the cost to the custodian. In Korea, companies such as Medipost, Histostem and Lifecode are actively doing business.

A common technique for recovering umbilical cord blood from a mother is to collect umbilical cord blood from umbilical blood vessels, place it in a sterile bag containing an anticoagulant, deliver it to a cord blood treatment and storage facility at room temperature, It is kept for up to 72 hours. To prevent blood clotting, sodium citrate, ACD solution (citrate dextrose), citrate phosphate dextrose (CPD), or CPDA-1 solution (CPD adenine-1) -1 blood bag is mainly used. The composition of the CPDA-1 solution was 0.299 g of anhydrous citric acid (molecular weight: 192.13), 2.63 g of sodium citrate (molecular weight: 294.10), 0.222 g of dihydrogenphosphate (molecular weight: 156.01), 3.19 g g (6-aminopurine, molecular weight 135).

The entire cord blood sample of the CPDA-1 blood bag that has arrived at the cord blood storage facility is aseptically transferred to a transfer bag, and then Hesplasma is added to increase plasma volume. Hess plasma is composed of 6.0 g of hydroxyethyl starch (molecular weight 130 Kd), 0.9 g of sodium chloride (molecular weight 58.5 Kd) and electrolyte (mEq / L), showing better plasma increasing effect than albumin or dextran. Hesplasma used for medical use increases the osmotic pressure in the plasma to show the effect of increasing the plasma, thus quickly restoring the cardiovascular function to normal. In addition, Hess plasma can be used safely because it has very little effect on blood coagulation and its structure is similar to glycogen in living body and does not cause antigen-antibody reaction. It can be safely used, and osmotic pressure almost similar to plasma can prevent destruction of blood cells by blood dilution, It has suitable pharmacological action and characteristics as a cyclic filling liquid.

Add Hess Plasma to the mobile bag, shake well, and centrifuge at 2500 rpm for 30 minutes at room temperature to remove red blood cells (RBCs) as much as possible without removing the mononuclear cell layers. The mononuclear cell layer from which erythrocytes have been removed is shaken again with the plasma layer, centrifuged at 2500 rpm for 30 minutes at room temperature, and the plasma is removed to the extent that the mononuclear cell layer is not removed together. After mixing the removed red blood cells and plasma layers, take some of them and separate the blood plasma and blood cells. They are used for blood type tests and virus tests. Then, the umbilical cord blood containing the plasma-depleted mononuclear cell layer is taken out from the mobile bag, mixed well with the freezing medium, frozen and stored in a liquid nitrogen tank.

In addition to the umbilical cord blood containing the mononuclear cell layer that is frozen as described above, the umbilical cord blood such as the red blood cells and the plasma layer remaining in the umbilical cord blood treatment and the umbilical cord blood mixture such as the anticoagulant and the plasma enhancer which have been previously added to the blood bag, It will be used for extrinsic fraud testing and the rest will be discarded. Therefore, efforts are needed to isolate useful components having physiological activity from such discarded cord blood wastes (CBWs).

The process of isolating serum for use as an additive for cell culture from animal blood is well known and fetal bovine serum (FBS) isolated from bovine fetus is the most widely used. FBS is usually obtained by freezing blood from fetuses, thawing the frozen blood to a certain volume, and then performing several stages of bacterial filtration. Animal serum contains various nutrients such as albumin, transport protein, hormone, growth factor, adhesion factor, amino acid and micronutrient, and is used as an additive for cell culture for the purpose of research and development for various purposes or industrially useful substances . However, animal sera are problematic due to the possibility of contamination with other unidentified cell growth harmful substances and bacteria, fungi, mycoplasma, viruses and mad cow disease factors. Although serum-free medium is used industrially in consideration of the problems of animal serum, it is still used in many fields due to the availability of serum [Valk J, Mellor D, Brands R, Fischer R, Gruber F, Gstraunthaler G, Hellebrekers L, Hyllner J, Jonker FH, Prieto P, Thalen M, Baumans V. (2004) The human blood collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicology in vitro 18: 1-1261].

Filtration and sterilization systems have been actively developed to remove adventitious contaminants in animal serum, but the complete removal of all contaminants has been difficult. The preparation of the bovine serum requires a strict quality control technique from the collection of the blood to the filtration and the charging. Generally, the filtration is performed three times using a filter having a pore size of 0.1 μm in the final stage of the production of the serum, A filter with a pore size of 0.04 μm at the final stage was prepared by a closed system collection method which can minimize the amount of endotoxin and hemoglobin in the serum such as Thermo Fisher of USA (Sigma, USA) is used in some products, or a step of filtering twice with a 0.2 탆 filter and then once with a 0.1 탆 filter is used. As such, animal-derived serum currently being marketed uses filtration to remove potential contaminants such as bacteria, fungi, mycoplasma, and viruses. In the final filtration step, most of the products except the hyclone product Serum manufacturers use 0.1 ㎛ filters at the final stage. Bacteria and fungi have a diameter of approximately 0.1 to 20 탆, and most of them are removed by a 0.1 탆 filter. However, since viruses have a diameter of 0.1 탆 or less, they are not completely removed by filtration of a 0.1 탆 filter, Of mycoplasma can pass through a filter of 0.1 mu m since it has no cell wall and has flexibility. That is, in the final step, the filtration using the 0.1 μm filter for 3 times can not completely remove the mycoplasma and the virus, and the filtration method using the 0.04 μm filter is performed in order to compensate for these disadvantages.

Human-derived blood plasma or blood serum can be readily separated from blood by well known methods such as centrifugation, sedimentation or filtration. Plasma typically refers to pale yellow liquid components in which the type components in the mammalian blood, cells and cell fractions, are separated, the components of which are well known in the literature. In the method of separating plasma from blood, centrifugation is performed at about 3,000 rpm for about 10 minutes to precipitate red blood cells, white blood cells, and cell fragments (platelets) and to separate the supernatant containing plasma.

Various methods have been used for separating useful components from human blood. For the purpose of treating specific diseases of the human body, specific fractions of human blood include cellular components (red blood cells and platelets) and plasma protein fractions (albumin, fibrinogen, globulin And blood factors) are increasingly used in the medical industry. In the field of human plasma fractionation technology, the most widely used plasma fractionation method for faster, higher yield, less denaturation and blocking of contamination is to collect frozen human plasma, thaw at below 5 ° C and centrifuge to remove precipitate (Avery CG, "Fractionation of Blood by Centrifugation.", Process Biochem . , 7.20, 1972), and a method of separating useful physiologically active substances by selectively precipitating plasma protein fractions (Curling JM , "Methods of Plasma Protein Fractionation, Ed. JM Curling, Academic Press, London UK, 1980).

In the case of a company aiming at the preservation of cord blood or stem cell storage business from umbilical cord blood of human origin, various contamination tests shall be conducted in order to guarantee the quality of all umbilical cord blood, which is the raw material, to prove that it is not pathogenic. Although there are reports that serum derived from human serum or umbilical cord blood can replace fatty acid serum, human serum is not only limited in the supply source but also has potential for viruses and other infectious diseases. Due to various reasons such as nonuniform composition, It is not used, but is used for a specific purpose only in a very limited study.

In the present invention, the biological umbilical cord blood mixture is subjected to a stepwise filtration system to efficiently remove the biological contaminants and foreign particles present in the human umbilical cord blood, and to recover the human umbilical cord blood- The present inventors have completed the present invention by confirming the cell growth ability in various animal cell lines of such low molecular weight fractions.

An object of the present invention is to provide a method for screening a cord blood (cord blood) containing an anticoagulant and a plasma enhancer, which is conventionally discarded after separating and storing a mononuclear cell layer (buffy coat) from cord blood collected from a mother for the purpose of cryopreservation of human umbilical cord blood And a method for fractionating a useful component having a physiological activity from the mixture by the molecular weight size.

It is another object of the present invention to provide a composition for nutritional supplementation of a cell culture medium derived from human and animal using various fractions obtained from a method for fractionating cord blood blood mixture.

In order to accomplish the above object, a method for fractionating a human umbilical cord blood mixture comprises centrifuging a cord blood blood mixture from which a mononuclear cell layer has been separated from umbilical cord blood, subjecting the mixture to sterilization filtration, performing ultrafiltration and nanofiltration so as to be filtered by molecular weight, .

Here, the umbilical cord blood mixture contains the anticoagulant and the plasma enhancer, meaning that the umbilical cord blood is taken from the mother into an aseptic bag containing the anticoagulant to sterilize the plasma enhancer and isolate the umbilical cord blood-derived mononuclear cell layer .

Preferably, the ultrafiltration and nanofiltration are performed at a filtration membrane size of 30 k Da and 3 k Da, respectively, and the filtration of the umbilical cord blood mixture finally separated is preferably a 0.1 탆 filter.

According to another aspect of the present invention, there is provided a composition for supplementing a culture medium of a cell culture medium, comprising a fraction obtained by separating a umbilical cord blood mixture from a umbilical cord blood, which has been separated from the umbilical cord blood, according to the molecular weight.

Here, the fraction of the umbilical cord blood mixture is selected from a fraction having a molecular weight size of 30 k Da or more, a fraction having a size of 3 k Da to 30 k Da, or a fraction having a molecular size of 3 k Da or less, and the composition for supplementing medium components of the cell culture medium containing such fraction Animal-derived serum, especially fetal bovine serum, can be substituted for the growth medium.

Hereinafter, the method and composition for fractionation of the umbilical cord blood mixture of the present invention will be described in detail.

Generally, in a company that aims to store umbilical cord blood, a cord blood is collected in a disposable sterile blood bag containing anticoagulant in advance for the purpose of cryopreservation of a specific cell layer derived from human cord blood, , And then sterilized by aseptically adding a plasma enhancer to collect only the mononuclear cell layer derived from the cord blood. The remaining umbilical cord blood mixture is usually discarded after collecting a part of the test sample.

In the present invention, in addition to the umbilical cord blood containing the mononuclear cell layer collected from the mother and thus frozen in this way, blood cells and plasma layer such as red blood cells remaining in the umbilical cord blood treatment process and anticoagulants and plasma enhancers previously added to the blood bags (CBW, cord blood wastes) by centrifugation, sterilization filtration, ultrafiltration, nanofiltration, and bactericidal filtration, and the resulting solution is finally filtered , And a fractionation process was performed using a nanofiltrated membrane of 3ka Da size so as to have a specific low molecular weight fraction. This fractionation process minimizes the amount of umbilical cord blood mixture that is practically discarded and is environmentally friendly. It is possible to take various kinds of physiologically active substances fractionated by various molecular weight sizes from the discarded umbilical cord blood mixture, and to carry out various research and industrial applications. Particularly, certain fractions obtained from the umbilical cord blood mixture discarded in accordance with the present invention are useful as animal serum substitutes which can replace bovine fetal serum used for culturing animal cells as well as human cells.

Specifically, a method for fractionating a human umbilical cord blood mixture according to the present invention comprises: centrifuging the umbilical cord blood mixture from which a mononuclear cell layer has been separated from umbilical cord blood at 2,500 rpm for 20 minutes to remove blood cell components (red blood cells and platelets) And then subjected to sterilization filtration using a 0.22 mu m filter to remove fine cell fragments or other solid matter. Next, the filtrate is filtered through an ultrafiltration membrane having a filtration membrane size of 30,000 daltons in the third order, and then subjected to a step filtration process using a nanofiltration filter having a filtration membrane size of 3,000 dalton (3,000 daltons). Finally, the obtained fractions are subjected to sterilization and filtration using a 0.1 mu m filter to obtain fractions of the umbilical cord blood mixture having a molecular weight size of 30 kDa or more, 3 kDa to 30 kDa, and 3 kDa or less.

Since the umbilical cord blood mixture used in the present invention is different in the amount of umbilical cord blood collected from the mother due to its characteristics, the volume to be collected is different for each individual, and since the ratio of the anticoagulant and the plasma enhancer are different among the whole blood mixture, The ratio of the anticoagulant to the plasma increase is different. In the present invention, the umbilical cord blood collected from each mother is prepared as an autologous serum of individual form, or two or more blood are classified according to ABO blood type, HLA form, or blood type or HLA.

In the case of a company aiming at the preservation of cord blood or stem cell storage business from umbilical cord blood of human origin, various contamination tests shall be conducted in order to guarantee the quality of all umbilical cord blood, which is the raw material, to prove that it is not pathogenic. Thus, all of the human umbilical blood used in the present invention were all non-reactive to hepatitis B surface antigen (HBsAg) and hepatitis C (HCV) antibodies and negative for HIV-1 and HIV-2 use.

That is, in the case of preserving the umbilical cord blood, since the umbilical cord blood mixture containing the various compounds to be separated from the umbilical cord blood is separated from the umbilical cord blood, the supply source of the umbilical cord blood to be discarded is of course limited. However, It is possible to confirm the safety as a raw material by inspecting various viruses (AIDS virus 1 and 2, hepatitis viruses B and C) and microorganisms (syphilis, bacteria and fungi) When the function as a nutrient having a physiologically active function is confirmed by fractionating a certain low molecular weight using the thus-secured raw material and finally sterilizing it by filtration, its utilization and value can be evaluated to be sufficiently high.

In addition, when fetal serum is supplementally added to the cell culture medium using the low molecular weight cord blood blood mixture fraction obtained according to the present invention, the fetal blood serum frequently used for various cell proliferation and cell survival improving activities is included Lt; RTI ID = 0.0 > and / or < / RTI > Thus, by replacing the fetal bovine serum added to the cell culture medium with the human umbilical cord blood fraction, contaminants originating from dangerous animals can be reduced or completely eliminated from the human body. In other words, using the abandoned umbilical cord blood mixture, which is a nutritional ingredient derived from the human body, will be economically advantageous as well as beneficial to the environment and commercially advantageous.

In addition, in the case of using the fraction of the umbilical cord blood mixture according to the present invention, the cord blood which is actually discarded is composed mainly of cellular blood and has an effect of reducing the blood waste amount by about 30% or less of the originally discarded volume.

According to the method of the present invention, it is possible to fractionate a useful component having physiological activity by size of molecular weight from the umbilical cord blood mixture which is usually discarded after storing cord blood, by the stepwise filtration. The thus- It is possible to obtain an effect of proliferating not only human-derived cells but also animal-derived cells by adding them as a supplement composition, and in particular, it is possible to reduce or eliminate the use of fetal bovine serum in a cell culture medium, The effect that can be expected is also expected.

Hereinafter, the present invention will be described more specifically by way of examples. However, these examples are only illustrative of the present invention, and the scope of the present invention is not limited to these examples.

Example  1: Containing anticoagulants and plasma enhancers Cord blood  Fractionation from the mixture by stepwise filtration

The umbilical cord blood mixture remaining after freezing and storing the mononuclear cell layer from the umbilical cord blood sampled from the mother is subjected to a stepwise filtration step including centrifugation, sterilization filtration, ultrafiltration, nanofiltration and bacteria elimination filtration, The components were fractionated by molecular weight size.

Since the amount of umbilical cord blood collected from a mother is different due to its characteristics, the amount of plasma enhancer added is also different. Thus, a cord blood blood mixture containing an anticoagulant and a plasma enhancer, which are separated from a cord blood and separated from a cord blood, are also different in volume. Also, because the ratio of anticoagulants to plasma elevators in the whole blood mixture is different, the ratio of the anticoagulant to the plasma elevator in the collected blood mixture is different. In this embodiment, the umbilical cord blood collected from each mother was prepared by combining autologous serum or two or more umbilical cord blood.

The umbilical cord blood mixture, in which the mononuclear cell layer was separated and discarded, was centrifuged at 2,500 rpm for 20 minutes to remove blood cell components (erythrocytes and platelets), and then subjected to a second filtration using a 0.22 쨉 m filter The solids were removed. The filtration was performed with an ultrafiltration membrane having a filtration membrane size of 30,000 daltons in the third order, and then subjected to nano-filtration stepwise filtration using a filter having a filtration membrane size of 3,000 dalton (3,000 daltons). Finally, each of the obtained fractions was subjected to sterilizing filtration using a 0.1 탆 filter to obtain a fraction having a molecular weight size of 30 k Da or more, 3 k Da to 30 k Da, and 3 k Da or less.

FIG. 1 is a schematic diagram showing a flow of a process of separating a mononuclear cell layer from cord blood collected from a mother for the purpose of preserving cord blood, and fractionating the umbilical cord blood mixture, which is normally disused, into a specific molecular weight size.

Table 1 shows the results of stepwise fractionation of three umbilical cord blood mixtures of different volumes. The results are shown in Table 1, which shows the change in the fractional volume obtained when the umbilical cord blood mixture samples prepared from different umbilical cord blood raw materials were subjected to the respective steps, The yield of fraction is shown. In Table 1, the mean values of the final volumes and the standard deviation of the fraction yields of the fractions obtained after the final filtering were 17.59 and 3.60 for fractions of 30 k Da or more, 7.89 and 1.03 for the fractions of 3 k Da to 30 k Da, respectively, The fractions below Da were 30.49 and 4.21, respectively. Although the volume of each umbilical cord blood differs depending on the characteristics of the umbilical cord blood, the volume of the umbilical cord blood mixture actually obtained varies depending on the volume of the plasma enhancer added. However, the volume of each final fraction obtained from the umbilical cord blood mixture solution has a standard deviation of 5 or less In general, the yield was stable.

step Fraction volume (ml) Fraction yield (%) Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Umbilical cord blood mixture solution 100.48 64.77 99.99 100.0 100.0 100.0 Centrifugation (2500 rpm, 15 min) 66.75 48.43 80.52 66.43 74.78 80.53 After 0.22 탆 sterilization filtration, the volume 66.35 46.94 79.07 65.04 72.48 79.07 Volume after 30k Da and 3K Da step filtration 60.85 45.93 73.59 60.58 70.93 73.60 0.1 ㎛ sterilization After filtration, volume 56.83 41.61 69.44 56.56 64.25 69.53   1) 30K daltons or more 18.18 10.98 23.61 18.10 16.97 23.69   2) Less than 30K daltons ~ 3K daltons or more  8.08  5.55 10.02  8.04  8.57 10.03   3) Less than 30K daltons 30.57 25.08 35.81 30.42 38.71 35.81

Example 2: Effect of umbilical cord blood fraction on CHO cell growth

The effect of the Chinese hamster ovary (CHO) - derived cell line on the growth of the umbilical cord blood fraction was investigated by comparing the growth in the medium containing the umbilical cord blood fraction.

(1) Growth of CHO cells in medium supplemented with umbilical cord blood fraction

CHO DG44 cells were inoculated into a protein-free medium supplemented with 5% concentration of each fraction obtained in Example 1 and a fetal calf serum (ThermoFisher, USA) and cultured in a floating culture flask (Erlenmeyer flask, Corning, USA). CHO DG44 cells were cultured in a cell line (CRL-9096 ™, CHO / dhfr-) purchased from the American Type Culture Collection (ATCC) in the USA using a serum-free medium (HyQSFM4CHO, USA, ThermoFisher) , ThermoFisher) in a floating culture system and then frozen. The frozen cell lines were thawed and cultured for the following experimental purposes. The culture was suspended at 37 ° C in a 5% CO ₂ incubator at 110 rpm on a suspension mixer, and the cell count and cell viability were measured every day.

(2) Method of measuring cell number

During cell culture, cell concentrations in cells and culture medium were periodically measured. The concentration of living cells was determined by trypan blue dye exclusion method. All measurements were repeated twice and the results were expressed as mean values.

(3) Effect of fractions on the growth of CHO cells

Fig. 2 shows the result of culturing for 8 days in each cell culture medium at a concentration of 5% for each fraction and a concentration of 5% for folate serum. 2A and 2B are graphs showing the degree of growth of a Chinese hamster ovary (CHO) -derived cell line in suspension culture in a cell culture medium supplemented with a umbilical cord blood mixture fractioned to a specific molecular weight, FIG. 2A is a graph showing the cell concentration, and FIG. 2B is a graph showing cell viability when compared with the growth in the culture medium.

As shown in FIG. 2A, CHO cell growth was increased in all fractions of umbilical cord blood mixture. In particular, the 30KS fraction (the first fraction) showed the highest cell growth, and the cell concentration in this fraction was similar to that of 5% fetal bovine serum. The cell viability of FIG. 2b was the best in the 30KF3KS fraction (the third fraction), the 30KS fraction (the first fraction) was similar to the 5% fetal calf serum, Slightly decreased but almost equal. The lowest cell viability is when the fourth fraction of 3KF is used.

Example  3: Cord blood  The mixture fraction Hybridoma  Effect on cell growth

The effect of umbilical cord blood fractionation on cell growth was investigated by comparing the extent to which hybridoma cells grew in the medium containing the umbilical cord blood fraction, compared to cell growth in medium containing fetal bovine serum.

(1) Growth of hybridoma cells in medium supplemented with umbilical cord blood fraction

Hybridoma cells (R4-6A2) were inoculated into the serum-free sterilized medium supplemented with 0%, 0.5%, 1%, 2.5%, and 5% concentrations of the fractions obtained in Example 1 and rabbit serum (ThermoFisher, USA) , And cultured in a floating culture flask (Erlenmeyer flask, Corning, USA). Hybridoma cells (R4-6A2) are monoclonal antibody-producing cell lines for mouse interferon gamma. They are distributed in the microbiology class at Kyunghee University Medical School and suspended in serum-free medium (HyQSFM4MAb, USA, ThermoFisher) After being adapted for culture, the cells were frozen and frozen cells were thawed and cultured for the following experimental purposes. The culture was suspended at 37 ° C in a 5% CO ₂ incubator at 110 rpm on a suspension mixer, and the cell count and cell viability were measured every day.

(2) Method of measuring cell number

During cell culture, cell concentrations in cells and culture medium were periodically measured. The concentration of living cells was determined by trypan blue dye exclusion method. All measurements were repeated twice and the results were expressed as mean values.

(3) Influence of fraction on the growth of hybridoma cells

The results obtained when the concentrations of the fractions were 0%, 0.5%, 1%, 2.5%, and 5%, and the concentrations of folate serum were 0%, 0.5%, 1%, 2.5%, and 5% Respectively. FIG. 3 shows the results of comparing the growth of hybridoma cells in a cell culture medium supplemented with various concentrations of umbilical cord blood fractions fractionated by a specific molecular weight, with growth of cell culture medium supplemented with fetal serum as a control It is a graph showing.

As shown in FIG. 3, cell growth was decreased with increasing concentrations of the umbilical cord blood mixture (Ori), the first fraction and the second fraction. In the 30KS3KF fraction (third fraction) and the 3KF fraction (fourth fraction) Cell growth was increased with increasing concentration. The increase in cell growth was highest in the 3KF fraction (fraction 4), which was similar or somewhat higher than that of 5% fetal bovine serum. In other words, it can be seen that the umbilical cord blood mixture has different effects on hybridoma cell growth depending on the fraction, and the 3KF fraction (fourth fraction) exhibits an excellent cell proliferation effect equivalent to or higher than that using the fetal serum.

Example  4: Cord blood  Effect of Mixture Fraction on MRC-5 Growth

The effect of umbilical cord blood fractionation on cell growth was assessed by comparing the extent to which human fetal lung cells (MRC-5) grew in medium containing umbilical cord blood fraction, compared to cell growth in medium containing fetal bovine serum Respectively.

Human fetal lung cells (MRC-5) were obtained from a cell line (CCL-171 ™) purchased from the American Type Culture Collection (ATCC) in the United States and cultured in DMEM medium supplemented with 5% fetal bovine serum (ThermoFisher, USA) (Tissue culture flask, Corning, USA), followed by cryopreservation. Cryopreserved cell lines were thawed and cultured for experimental purposes. The culture medium was DMEM medium (high glucose). MRC-5 cells were cultured for 7 days at 5% and 10% concentrations of the fetal calf serum and umbilical cord blood mixture, respectively. On the 5th and 7th days, Were harvested and cell numbers were measured and cell growth was compared.

4A and 4B are graphs showing the degree of growth of the human lung cell line (MRC-5) in the cell culture medium added with the kind and concentration of the umbilical cord blood mixture fractionated by the specific molecular weight size, FIG. 4A shows the number of viable cells at 5% addition, and FIG. 4B shows the number of viable cells at 10% addition.

As shown in FIGS. 4A and 4B, the degree of cell growth in all the cell culture media was similar on the fifth and seventh days of culture. (30KS) and the third fraction (30KF3KS) showed slightly lower cell growth of about 10%, compared with the cell growth of fetal calf serum at each concentration. However, the crude fraction and the second fraction (30KF) The fraction (3KF) showed very low cell growth around 30%. These results show that the effects of different types of umbilical cord blood fraction on the growth of human fetal lung cells are different, with no significant difference in the concentration between 5% and 10%. In conclusion, it can be shown that using the appropriate fraction of umbilical cord blood mixture at an appropriate concentration can replace fetal bovine serum for the growth of human fetal lung cells. For example, the first or third fraction of umbilical cord blood mixture It can be used at a concentration of about 5%.

5 is a photomicrograph (magnification 100 times) showing the shape of a human lung cell line (MRC-5) when cultured for 5 days in a cell culture medium containing 5% concentration of umbilical cord blood mixture fraction and fetal bovine serum. 5, the cell density was highest in the 5% FBS culture, and the cell growth was slightly lower in the first fraction (30KS) and the third fraction (30KF3KS) than in the other cells. Which is almost the same as the culture used. From the above results, it can be seen that all fractions of the umbilical cord blood mixture do not change the shape of human fetal lung cells.

FIG. 1 is a schematic diagram showing a flow of a process of separating a mononuclear cell layer from cord blood collected from a mother for the purpose of preserving cord blood, and fractionating the umbilical cord blood mixture, which is normally disused, into a specific molecular weight size.

2A and 2B are graphs showing the degree of growth of a Chinese hamster ovary (CHO) -derived cell line in suspension culture in a cell culture medium supplemented with a umbilical cord blood mixture fractioned to a specific molecular weight, FIG. 2A is a graph showing the cell concentration, and FIG. 2B is a graph showing cell viability when compared with the growth in the culture medium.

FIG. 3 shows the results of comparing the growth of hybridoma cells in a cell culture medium supplemented with various concentrations of umbilical cord blood fractions fractionated by a specific molecular weight, with growth of cell culture medium supplemented with fetal serum as a control It is a graph showing.

4A and 4B are graphs showing the degree of growth of the human lung cell line (MRC-5) in the cell culture medium added with the kind and concentration of the umbilical cord blood mixture fractionated by the specific molecular weight size, FIG. 4A shows the number of viable cells at 5% addition, and FIG. 4B shows the number of viable cells at 10% addition.

5 is a micrograph (magnification 10 times) showing the shape of a human lung cell line (MRC-5) when cultured for 5 days in a cell culture medium containing 5% concentration of umbilical cord blood mixture fraction and fetal bovine serum.

Claims (7)

The umbilical cord blood mixture from which the mononuclear cell layer was separated from the umbilical cord blood was centrifuged, filtered, and subjected to ultrafiltration and nano-filtration so as to be filtered with a fraction having a molecular weight of 30 k Da or more, a fraction having 3 k Da to 30 k Da, 30 k Da, and 3 k Da, and subjecting the mixture to filtration for sterilization. The method according to claim 1, wherein the umbilical cord blood mixture comprises an anticoagulant and a plasma enhancer. delete The fraction of umbilical cord blood mixture from which the mononuclear cell layer has been separated from cord blood comprises a fraction selected from a fraction having a molecular weight size of 30 kD or more, a fraction of 3 k Da to 30 k Da, or a fraction of 3 k Da or smaller. A composition for supplementing a culture medium of a cell culture medium. delete 5. The composition for supplementing a nutrient composition according to claim 4, which is for replacing an animal-derived serum in a cell culture medium. The composition of claim 4, wherein the fetal bovine serum is replaced with a cell culture medium.

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