Preparation method of concentrated red blood cells from umbilical cord blood

Abstract

The invention discloses a preparation method of concentrated red blood cells from umbilical cord blood, and belongs to the field of biomedical engineering.According to the technical scheme, the preparation method comprises the following steps that umbilical cord blood is collected, whole umbilical cord blood is directly connected with a triple bag and then subjected to 600 * g centrifugation for 7 min at the temperature of 4 DEG C, umbilical cord blood plasma, umbilical cord blood mononuclear cells and suspended red blood cells are separated out, and the umbilical cord blood mononuclear cells and the suspended red blood cells are collected; and continuously centrifuging the obtained suspension red blood cells at 4 DEG C for 7min by 5000 * g, further concentrating the red blood cells, and removing upper plasma to obtain the concentrated red blood cells of the umbilical cord blood. According to the invention, 6% hydroxyethyl starch which is conventionally used is not added during preparation, but after technical optimization, aggregation and precipitation of red blood cells due to structural change are avoided, and the oxygen storage and transportation functions of the red blood cells are reserved, so that the red blood cells can be used as a red blood cell resource source. The hematocrit of the concentrated red blood cells obtained by the method is higher than 80%, the hemoglobin content recovery rate is higher than 55%, and the concentrated red blood cells can be used for autologous retransfusion of red blood cells during anemia of neonates, especially premature infants.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering and relates to the preparation of blood products, and in particular to a method for preparing concentrated red blood cells derived from umbilical cord blood. Background Art

[0002] Anemia in newborns is a common complication, especially in premature infants. Premature infants have immature systems, including the hematopoietic system, which may lead to anemia. Anemia seriously affects the growth and development of premature infants and may lead to other complications, even life-threatening. Transfusion of concentrated red blood cells is a routine treatment. Most premature infants with a gestational age of ≤32 weeks will receive more than two transfusions two weeks after birth. Adult red blood cells are transfused, and the amount of each transfusion is less than 50mL based on the weight of the newborn. For newborns and premature infants, because adult red blood cells are highly differentiated and immunogenic, allogeneic transfusions are prone to adverse reactions, including fever, urticaria, hemolysis, viral infection and immunosuppression, and low transfusion volume is also prone to blood waste. Umbilical cord blood is blood rich in stem cells and red blood cells collected from the umbilical cord of a newborn at birth. If used as a source of autologous transfusion blood, it has the advantages of being available at any time and without immune rejection, which can greatly avoid the above-mentioned adverse reactions and blood waste of allogeneic transfusions.

[0003] Umbilical cord blood is a precious biological resource, which contains a variety of stem cells such as hematopoietic stem cells and endothelial progenitor cells. In the field of biomedical engineering, umbilical cord blood has been widely used in clinical treatment, especially in hematopoietic stem cell transplantation. It can replace the source of bone marrow hematopoietic stem cells and is used for transplantation of blood diseases in children and adults. The umbilical cord blood currently used in clinical practice specifically refers to umbilical cord blood mononuclear cells, that is, the cell group (buffy coat) after separating plasma and red blood cells. When preparing umbilical cord blood mononuclear cells, umbilical cord blood banks usually add 6% hydroxyethyl starch to make the red blood cells form a "coin string" structure to aggregate and precipitate, so as to separate and precipitate the red blood cells, thereby obtaining high-purity mononuclear cells. The separated and precipitated red blood cells lose their function due to structural changes and will be discarded, wasting the resource of umbilical cord blood red blood cells. However, umbilical cord blood-derived red blood cells are also a precious resource and can be used as a reserve resource for autologous blood transfusion in premature infants. It is urgent to develop a method for preparing and storing umbilical cord blood red blood cells suitable for newborns and premature infants. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a method for preparing concentrated red blood cells from umbilical cord blood. Through technical optimization, 6% hydroxyethyl starch is not added in the existing preparation process of umbilical cord blood mononuclear cells. Without affecting the original preparation of umbilical cord blood mononuclear cells, the red blood cells are not aggregated and precipitated, and lose the oxygen transport ability and are discarded. Instead, the red blood cell structure and oxygen transport function are retained, and then centrifuged to prepare concentrated red blood cells, so that they can be used as a source of red blood cell resources for autologous red blood cell transfusion in newborns, especially premature infants with anemia.

[0005] The specific scheme adopted by the present invention is:

[0006] A method for preparing concentrated red blood cells from umbilical cord blood comprises pre-treatment of the umbilical cord blood, separation of mononuclear cells and red blood cells, and preparation of concentrated red blood cells by centrifugation; specifically comprises the following steps: Step 1: Collect whole umbilical cord blood into a blood collection bag containing CPDA-1 blood preservation solution, shake well, seal the tube with a heat sealer, and transport it to the GMP production workshop for preparation; Step 2: Connect the whole umbilical cord blood to a triple bag and centrifuge at 600×g for 7 min at 4°C to separate the three bags of products: umbilical cord plasma, umbilical cord blood mononuclear cells and suspended red blood cells; add a cryoprotectant to the obtained umbilical cord blood mononuclear cells for freezing; Step 3: Centrifuge the suspended red blood cells obtained in step 2 at 5000×g for 7 min at 4°C, and remove the upper plasma to obtain concentrated red blood cells from the umbilical cord blood.

[0007] As a further optimization of the above preparation method, the CPDA-1 blood preservation solution is added when collecting whole cord blood and used as an anticoagulant and storage solution.

[0008] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0009] (1) Preventing red blood cells from aggregating and precipitating and losing their oxygen transport function: In the prior art, when preparing red blood cells from umbilical cord blood, 6% hydroxyethyl starch needs to be added to separate and precipitate red blood cells, which will cause the red blood cells to aggregate and precipitate due to structural changes, affecting the red blood cells' oxygen transport function and requiring disposal. The present invention adopts a unique cord blood pretreatment method, which does not add 6% hydroxyethyl starch to the whole blood of the umbilical cord blood, thereby preventing the red blood cells from aggregating and precipitating due to structural changes, thereby retaining the red blood cells' oxygen transport capacity and making it a source of red blood cells.

[0010] (2) The method for preparing red blood cells from umbilical cord blood of the present invention does not affect the preparation of umbilical cord blood mononuclear cells. This method not only simplifies the preparation process and improves the preparation efficiency, but also avoids the side effects and risks that may be caused by adding chemical reagents.

[0011] (3) Improving the purity and use effect of red blood cells: This technical solution optimizes the centrifugation and separation method. After the umbilical cord blood mononuclear cells and suspended red blood cells are obtained by the initial centrifugation, the suspended red blood cells are centrifuged again to further concentrate the red blood cells, thereby improving the quality and purity of the concentrated red blood cells obtained from the umbilical cord blood, making the hematocrit higher than 80%, and the hemoglobin content recovery rate higher than 55%, thereby improving the purity and use effect of the red blood cells.

[0012] (4) Developing a method for preparing and storing umbilical cord blood red blood cells suitable for newborns and premature infants: Existing technologies are mainly aimed at adult red blood cells. However, for newborns and premature infants, due to the particularity of their hematopoietic system and immune system, the use of adult red blood cells for transfusion may cause adverse reactions. The present technical scheme provides a method for preparing and storing autologous umbilical cord blood red blood cells suitable for newborns and premature infants, avoiding adverse reactions such as fever, urticaria, hemolysis, viral infection and immunosuppression that may be caused by allogeneic blood transfusion, while also avoiding blood waste caused by low transfusion volume, and has important clinical application value. In general, compared with the existing technology, the present invention provides a safer, more efficient and more practical method for preparing red blood cells derived from umbilical cord blood, which has significant advantages. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0014] Unless otherwise specified, the methods used in the present invention are all conventional technical means, and the raw materials, reagents, equipment, etc. used are all conventionally available on the market.

[0015] Example 1: Step 1: Collect the whole blood of the umbilical cord blood of the newborn into a blood collection bag containing CPDA-1 blood preservation solution, shake well, seal the tube with a heat sealer, and transport it to the GMP production workshop for preparation. In the specific operation, the collection volume of the whole blood of the umbilical cord blood is 88mL, and the amount of CPDA-1 blood preservation solution added is 1 / 10 of the volume of the whole blood of the umbilical cord blood. Step 2: After connecting the whole blood of the umbilical cord blood to the triple bag, centrifuge it at 600×g for 7 minutes at 4°C to prepare the umbilical cord blood mononuclear cells and separate the suspended red blood cells, and separate the three bags of umbilical cord plasma, umbilical cord blood mononuclear cells and suspended red blood cells. In the specific operation, keep the temperature constant, the speed stable, and the time accurate during the centrifugation process. Step 3: Add a cryoprotectant to the obtained umbilical cord blood mononuclear cells for freezing. In the specific operation, the amount of cryoprotectant added is 1 / 10 of the volume of the umbilical cord blood mononuclear cells, and the cryoprotectant uses DMSO (dimethyl sulfoxide). Step 4: The suspended red blood cells are centrifuged at 5000×g for 7 minutes at 4°C, and the upper plasma is removed to obtain concentrated red blood cells from umbilical cord blood. In the specific operation, the temperature is kept constant, the rotation speed is stable, and the time is accurate during the centrifugation process. Step 5: The volume, hematocrit, and hemoglobin content recovery rate of the concentrated red blood cells from umbilical cord blood are measured. In the specific operation, the hematocrit and hemoglobin content are measured using a Sysmex blood cell counter. Through the above steps, the hematocrit of the concentrated umbilical cord blood red blood cells obtained is 90.4%, and the hemoglobin content recovery rate is 58.7%.

[0016] Example 2: Step 1: Collect the whole blood of the umbilical cord blood of the newborn into a blood collection bag containing CPDA-1 blood preservation solution, shake well, seal the tube with a heat sealer, and transport it to the GMP production workshop for preparation. In the specific operation, the collection volume of the whole blood of the umbilical cord blood is controlled at 50-100 ml, and the amount of CPDA-1 blood preservation solution added is 1 / 10 of the volume of the whole blood of the umbilical cord blood. Step 2: After connecting the whole blood of the umbilical cord blood to the triple bag, centrifuge it at 600×g for 7 minutes at 4°C to prepare the umbilical cord blood mononuclear cells and separate the suspended red blood cells, and separate the three bags of umbilical cord plasma, umbilical cord blood mononuclear cells and suspended red blood cells. In the specific operation, the temperature is kept constant, the speed is stable, and the time is accurate during the centrifugation process. Step 3: The obtained umbilical cord blood mononuclear cells are added with a cryoprotectant for freezing. In the specific operation, the amount of cryoprotectant added is 1 / 10 of the volume of the umbilical cord blood mononuclear cells, and the cryoprotectant uses DMSO (dimethyl sulfoxide). Step 4: The suspended red blood cells are centrifuged at 5000×g for 7 minutes at 4°C, and the upper plasma is removed to obtain concentrated red blood cells from umbilical cord blood. In the specific operation, the temperature is kept constant, the rotation speed is stable, and the time is accurate during the centrifugation process. Step 5: The hematocrit and hemoglobin content recovery rate of the concentrated red blood cells from umbilical cord blood are measured. In the specific operation, the hematocrit and hemoglobin content are measured using a Sysmex blood cell counter. Through the above steps, the hematocrit of the concentrated red blood cells obtained is 89.2%, and the hemoglobin content recovery rate is 69.2%.

[0017] Example 3: Step 1: Collect the whole blood of the umbilical cord blood of the newborn into a blood collection bag containing CPDA-1 blood preservation solution, shake well, seal the tube with a heat sealer, and transport it to the GMP production workshop for preparation. In the specific operation, the collection volume of the whole blood of the umbilical cord blood is controlled at 50-100 ml, and the amount of CPDA-1 blood preservation solution added is 1 / 10 of the volume of the whole blood of the umbilical cord blood. Step 2: After connecting the whole blood of the umbilical cord blood to the triple bag, centrifuge it at 600×g for 7 minutes at 4°C to prepare the umbilical cord blood mononuclear cells and separate the suspended red blood cells, and separate the three bags of umbilical cord plasma, umbilical cord blood mononuclear cells and suspended red blood cells. In the specific operation, keep the temperature constant, the speed stable, and the time accurate during the centrifugation process. Step 3: Add a cryoprotectant to the obtained umbilical cord blood mononuclear cells for freezing. In the specific operation, the amount of cryoprotectant added is 1 / 10 of the volume of the umbilical cord blood mononuclear cells, and the cryoprotectant uses DMSO (dimethyl sulfoxide). Step 4: The suspended red blood cells are centrifuged at 5000×g for 7 minutes at 4°C, and the upper plasma is removed to obtain concentrated red blood cells from umbilical cord blood. In the specific operation, the temperature is kept constant, the rotation speed is stable, and the time is accurate during the centrifugation process. Step 5: The hematocrit and hemoglobin content recovery rate of the concentrated red blood cells from umbilical cord blood are measured. In the specific operation, the hematocrit and hemoglobin content are measured using a Sysmex blood cell counter. Through the above steps, the hematocrit of the concentrated red blood cells obtained is 91.6%, and the hemoglobin content recovery rate is 56.5%.

[0018] The present invention can be widely used in the application fields such as biomedical engineering and blood product preparation. First, in the field of biomedical engineering, the method for preparing red blood cells derived from umbilical cord blood of the present invention provides a new idea for the utilization of umbilical cord blood. Through the method of the present invention, high-quality umbilical cord blood red blood cells can be obtained, which will greatly promote the research and application in the field of biomedical engineering, especially in transfusion therapy and the like, and is expected to improve the effect and success rate of treatment. Secondly, in the field of blood product preparation, the method for preparing red blood cells derived from umbilical cord blood of the present invention provides a new technical route for the preparation of blood products. The traditional preparation method requires the addition of 6% hydroxyethyl starch, which may affect the purity and quality of red blood cells. The method of the present invention avoids this step and can directly obtain high-purity red blood cells, which will greatly improve the preparation efficiency and quality of blood products and meet the market demand for high-quality blood products. In general, the method for preparing red blood cells derived from umbilical cord blood of the present invention has broad application prospects and market demand, and is expected to play an important role in the application fields such as biomedical engineering and blood product preparation.

[0019] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A method for preparing concentrated red blood cells from umbilical cord blood, comprising pre-treatment of the umbilical cord blood, separation of mononuclear cells and red blood cells, and preparation of concentrated red blood cells by centrifugation; characterized in that: The specific steps include: Step 1: Collect whole umbilical cord blood into a blood collection bag containing CPDA-1 blood preservation solution, shake well, seal the tube with a heat sealer, and transport it to the GMP production workshop for preparation; Step 2: Connect the whole umbilical cord blood to a triple bag and centrifuge at 600×g for 7 min at 4°C to separate the three bags of products: umbilical cord plasma, umbilical cord blood mononuclear cells and suspended red blood cells; add a cryoprotectant to the obtained umbilical cord blood mononuclear cells for freezing; Step 3: Centrifuge the suspended red blood cells obtained in step 2 at 5000×g for 7 min at 4°C, and remove the upper plasma to obtain concentrated red blood cells from the umbilical cord blood.

2. The method for preparing concentrated red blood cells from umbilical cord blood according to claim 1, characterized in that: The CPDA-1 blood preservation solution is added when collecting whole cord blood and is used as an anticoagulant and storage solution.

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