JPH07500597A - How to freeze cells for transplantation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to freeze cells for transplantation The present invention relates generally to methods of freezing cells, and more specifically to methods of freezing cells for transplantation. The present invention relates to a method of freezing cells (grafting).

Background of the invention Bone marrow transplantation (BMT) is a special treatment for patients with certain tumor or blood disorders ( Kamni et al., “Bone marrow transplanta” tion, Ploblems and prospec Koto A” Med C11n, North, Amer, 68:657-674.19 85). For cancer treatment, the doses of conventional cytotoxic drugs and radiotherapy include: There are limitations due to toxicity to bone marrow stem cells.

To overcome this difficulty, treat patients with high-dose chemotherapy and radiation therapy. After that, allogeneic bone marrow transplantation (BMT) was performed as a support. allogeneic bone marrow Transplants significantly improve survival in patients with aplastic anemia, acute leukemia, and severe immunodeficiency. However, many difficulties have limited its clinical application. J, most Which patients have a histocompatible sibling donor are generally amenable to such a transplant. It all depends on finding the right toner. Second, the reception Patients must be immunosuppressed before transplantation. Third, allogeneic bone marrow transplantation Severe complications caused by plant-versus-host disease and infection can occur.

Compared to allogeneic bone marrow transplantation, autologous bone marrow transplantation (ABMT) has considerable advantages. There is. It is a disease that has nothing to do with the bone marrow, such as malignant lymphoma or melanoma. , and patients with a variety of solid tumors, including lung and breast carcinomas. It has been used in combination with strong chemotherapeutics for treatment. ABMT itself is a direct tumor Chemotherapy and radiation that do not act on cells but are stronger due to the regeneration of immune and hematopoietic activity can facilitate treatment (Gorin et al., “Chemoth erapy and out.

1ogous bone Mrrow Transplantation in Acute leukemias, malignant Geki Takadare ■Shield■ as and 5 solid jamors, -Eur, J, Cancer 17:557-568.1981). Recently, ABMT has been leukemias, including lymphocytic and nonlymphocytic leukemias, and chronic granular leukemia in the blast stage; as commonly used to treat patients with lymphoma and breast cancer, as well as patients with lymphoma and breast cancer. It has become.

The benefits associated with bone marrow repopulation after chemotherapy or radiation therapy may help preserve the bone marrow. existence has received considerable scientific attention. However, in order to obtain the benefits of ABMT, For this purpose, bone marrow must be stored for a period of time to allow for chemotherapy or radiation treatment of the patient. Must be. Early studies on bone marrow storage showed that storage at room temperature or 4°C It has been shown that rapid loss of transplantable cells results. Thus, bone marrow autologous If a blood transfusion is delayed for more than a few days, bone marrow may not be able to be retransplanted into the patient. be. Complications sufficient to delay autotransfusion are common among patients with advanced cancer. This is the reason why this storage method is not supported. After that, several people Scientists at It was shown that the blood could be successfully thawed and autotransfused into patients.

However, one major problem with current methods is that bone marrow cannot be transplanted or repopulated. The reason is that only a relatively small percentage of real-world cells survive this cryopreservation method. Ru. For example, Lemori et al. 73:101-104.1988) collected whole bone marrow and separated the mononuclear fraction.

These cells were frozen, but 50-66% of CFU-G~1 cells were recovered by thawing. It was only possible. Figuera et al. (Cryobiol ogy 23:470-475.1986) is stored using a rate-controlled refrigeration system. Although the bone marrow buffy coat was frozen in the bag. Approximately 36-43% of CFU-GM cells were recovered. It was only a matter of time. Pisa et al., Cryobiology 20:587-590 (1983) cooled the bone marrow pia mater in a rate controlled refrigerator. Only about 70% of the cells were recovered after freezing and cryopreservation.

Additionally, current cryopreservation methods typically require large amounts of cells to be protected during freezing and thawing. Use DMSO. For example, patients receiving partial bone marrow transplants typically receive 10 to A patient who receives 20 ml of DMSO and receives whole bone marrow will receive 150 ml of DMSO. SO may be administered.

Patients already weakened by chemotherapy or radiation therapy can be treated with large doses of DMSO. May exhibit toxic effects. Patients receiving large doses of DMSO may experience unpleasant breath and In addition to experiencing the smell, you may also experience headaches, nausea, chills, dizziness, and vomiting. There are many. Additionally, more serious complications include arrhythmia, eye damage, high blood pressure, kidney failure, and even death. There is also the risk of problems.

Finally, current methods of cryopreservation of bone marrow allow cell agglutination and cell lysis of red blood cells. often occurs. Hemoglycemia released by these aggregates and lysed red blood cells Robin is suffering from deep pulmonary stress and kidney failure. (related and occasionally seen in bone marrow recipients).

The present invention addresses the toxic effects of DMSO, cell clumps and lysed red blood cells, and prior art refrigeration. It overcomes the poor cell recovery rate of cryopreservation methods and also provides other related benefits.

Summary of the invention Briefly stated, the purpose of the present invention is to provide a method for preparing cells for transplantation for future use. It is to provide law. More specifically, cells for transplantation are prepared and frozen for long periods of time. , which can later be put back together for treatment or research.

In one aspect of the invention, (a) cells for transplantation are purified from a suitable blood product; (b) concentrating the purified cells for transplant; (C) freezing and thawing the concentrated cells; Approximately 10XIO' to 40XIO' in a solution that substantially maintains cell viability during freezing. (d) the resuspended cells were tested for cell viability. future use of the cells for transplantation, including freezing under a first set of conditions to substantially maintain A method is provided for preparing. In a related aspect of the invention, (a ) purifying cells for transplantation from an appropriate blood product, and (b) concentrating the purified cells for transplantation. (C) subjecting the enriched cells to substantially maintaining cell viability during freezing and thawing; resuspend in a solution containing a total of about 0.002 to about 1 ml of DMSO. and (d) subjecting the resuspended cells to a first solution that substantially maintains cell viability. A method of preparing cells for transplantation for future use, including the step of freezing them under standard conditions. law is provided.

In one embodiment of the invention, the purified cells for transplantation are about 10XIO' to about 40XIO' Resuspend at a concentration of XIO'' cells/ml.

In one embodiment, the method described above determines cell viability following a freezing step. The method further includes thawing under a second set of conditions that maintain substantially the same. In a preferred embodiment In the next step, the cells were thawed in a 37°C water bath and then slowly washed with physiological buffer in a subsequent step. dilute.

In other embodiments of the invention, the purification step is performed using an immunoaffinity method for purifying cells for transplantation. The concentration step involves passing the blood product through a tic column and centrifuging the cells for transplantation. Including.

In other embodiments, the enriched cells are placed in a solution containing about 6% HES, or all in a solution containing about 0.002 to about 1 ml of DMSO, or preferably, culture medium. , protein, and an osmotic cryoprotectant. particularly preferred The culture medium includes RPMII640, TCI 99, and Iscove' s) Contains DMEM.

In yet another embodiment, the freezing step comprises freezing the cells at a controlled rate. This is done by In a currently preferred embodiment, (a) cooling the cells to about 4°C; (b) the cells at 4°C are heated at a rate of about -4°C until the cells reach about -4°C; Cooling: (C) the -4°C cells at about 0.5°C until the cells reach about -20°C; (d) cooling the -20°C cells until the cells reach about -40°C; and (e) cooling the -40°C cells at a rate of about 1.0°C/min to control by cooling at a rate of about 10.0°C/min until it reaches about -90°C. Perform the freezing process at the same speed.

Another aspect of the invention provides: (a) a therapeutic amount of implantable tissue prepared according to the invention; and (b) a total of about 0.002 to about 1 ml of DMSO. A composition is provided. In one embodiment, administering such a composition to a patient A method of treating an immunocompromised patient is provided. Administration is this Can be performed using either a syringe or an IV bag containing the composition .

These and other aspects of the invention will be apparent to those skilled in the art with reference to the following detailed description and accompanying drawings. will be obvious.

Brief description of the drawing FIG. 1 is a graph illustrating the freezing of cells for transplantation at a controlled rate.

Figure 2 shows the viability, cell recovery rate, and colony shape of cryopreserved purified cells for transplantation. FIG. 2 is a bar graph comparing the recovery rate of adult cells (CFC) with cryopreserved whole bone marrow.

Figure 3 shows the viability and details of purified cells for transplantation that were frozen and cryopreserved at different cell concentrations. It is a bar graph comparing cell recovery rate and CFC recovery rate.

Figure 4 shows purified transplant cells stored frozen in 7.5% DMSO and 10% DMSO. 1 is a bar graph comparing cell viability, cell recovery rate, and CFC recovery rate.

Detailed description of the invention As indicated above, the present invention provides a method for preparing cells for transplantation for future use. provide. Within the context of the present invention, the term "cells for transplantation" includes totipotent hematopoietic stem cells , as well as early progenitor cells such as colony forming cells (CFCs). C.F.C. Typical examples include CFU-E, CFU-G, CFU-M, CFU-GM, CFU-G Includes EMM, and BFU-E cells. Generally, 1/3 to 1/3 of CD34 positive cells Due to the fact that only 2/3 can be stem cells or colony forming cells, C When D34 cells are concentrated or purified, cells for transplantation must be concentrated or purified to the same extent. It is considered necessary to do so.

Utilizing the devices and methods described in more detail below, peripheral blood and whole bone Cells for transplantation can be purified from a variety of blood products, including marrow. For the present invention If at least 20% of the purified cells are CD34 positive cells, The cells are considered to be "purified," preferably containing 90% of CD34 positive cells. should be purified to a higher purity. Furthermore, the recovery rate and viability of cells for transplantation Reduces platelets, granulocytes, and red blood cells to prevent aggregation and release of degrading enzymes It is desirable to keep the total number of balls as low as possible. More specifically, purified transplantable less than about 1% platelets, less than 50% and preferably less than about 25% granulocytes; and generally contain less than 10% and preferably less than about 1% red blood cells. desirable.

Cells for transplantation are purified using a ligand that specifically recognizes the antigen on these cells. It can be carried out. For example, to purify cells for transplantation, specific may be used in the devices and methods described below. CD34 antigen Representative examples of antibodies that specifically recognize MY-10 and HPCA2 (Bect on-Dickinson, Mountain View, Calif, ), QBEND-10 (Quantul Biosystems.

Cambridge, U, Ni,), and 12.8 (CellPro ■, B othell, Wash,).

Magnetic beads, panning, and flow cytometry (fluorescence-activated cell separation and collection) Purification of cells for transplantation using a variety of methods and devices, including the use of (e.g., U.S. Pat. Nos. 4,714,680 and 4,965,2) No. 04, the contents of which are hereby incorporated by reference). Especially good A preferred method and device is “Inn+unselection Device”. U.S. Patent Application No. 071,513.543 entitled The contents of the specifications shall be incorporated herein by reference. It is an affinity column. Briefly, this application applies to transplantable cells such as cells for transplantation. Method and method for isolating or separating target particles from a mixture of non-target particles and target particles The equipment is described. This application includes a ligand that can specifically bind to target particles. By passing the particles through a column containing a layer of porous material with low non-specific binding, It includes a discussion of devices and methods for separating target particles by direct methods. This out In one aspect of the application, the invention generally includes (a) an inlet through which liquid can enter the column; and a distal end having an outlet through which liquid can exit the column. (b) a layer of porous material with low non-specific binding within the column; A porous substance has a biotin-adsorbing group immobilized on its surface, and the porous substance The pores of the layer are large enough for the biotin-adsorbing group to fit therein, but the layer is The space between the layers is not large enough to cause particles to pass through the layer. A device is provided that includes a layer that is large enough to flow. The device means disposed within the porous material for agitating the porous material by applying an external force; The method may include means by which bound target particles are released from the porous material. child In other aspects of the application, steps 1 or 2 using avidin and biotin are described. Separate the target particles by any of the step methods.

However, for the purposes of this application, other materials, including non-porous materials, may be Can be used in affinity columns.

A particularly preferred immunoaffinity membrane is “Improved Appara tus and Method for Ce1l 5eparation” (attorney's docket number 200072.407) The contents of this document shall be incorporated herein by reference. To explain briefly , in one aspect of this application, for separating target cells from a sample liquid. “Cell separation device (call 5 eparator)” including column assembly is provided. and the column assembly includes a column, a sample liquid supply bag, and a liquid collection bag. nothing. In this case, the column receives sample liquid from a sample liquid supply bag; and is equipped to separate target cells from the sample liquid and retain the target cells. and the fluid collection bag is adapted to receive target cells after they are released from the column. Sea urchins are prepared. The cell separation device stirs the contents of the column and maintains it within the column. an agitation means that aids in the release of retained sample cells; the amount of agitation of the column contents; a means of altering the release rate of sample cells in response to a drive signal that alters the , the column signal indicates the optical density of the liquid flowing from the column into the liquid collection bag. Column sensor means for supplying fluid as it exits the column and into the fluid collection bag. Responds to column valve control signals to selectively enable flow column valve means for controlling the cell separation device, and a device for controlling the operation of the cell separation device. A data processor that controls drive signals and column valves. Steps to deliver signals and prevent collection of inappropriate concentrations of target cells Including steps. One aspect of this invention is Ce1lPro@(Bothell, W CEPRATE LC™ Cell Separation System available from Ru.

Next, the purified cells for transplantation are concentrated. Various methods including, for example, sedimentation and filtration Although purified cells for transplantation can be concentrated using Yes. In a preferred embodiment of the invention, centrifugation at 150Xg for 10 minutes Concentrate the purified cells for transplantation and discard the supernatant.

The explants are then concentrated in a solution that substantially maintains cell viability during freezing and thawing. Resuspend the cells for use. In the context of the present invention, more than 80% of the cells, preferably Cell viability is defined as if more than about 90% of the cells are viable after freezing and thawing. “Substantially maintained.” Particularly preferred method for measuring the viability of cells for transplantation The method is described in more detail in Example 8 below.

Using various solutions to substantially maintain cell viability during freezing and thawing Can be done. These solutions prevent or reduce the formation of intracellular crystals (],), and (2) ) increase intracellular osmotic pressure to reduce volume loss during freezing, and (3) stabilize cell membranes. Maintain cell viability during freezing and thawing by stabilizing the cells. The main component of the solution Ingredients may be, for example, plain saline or buffered saline (e.g., saline prephosphate). buffer or “PBS”), or any physiologically acceptable medium, including cell culture media. It may also consist of a liquid. Representative examples of cell culture media suitable for use in the present invention contains R, PMr l 640, TCl 99, or Iscot D ME M (Gibco) BRI, Gaithersburg, M d, available from ).

The solution may further contain an osmotic cryoprotectant, a membrane stabilizer, a protein, or preferably three It should contain all substances. such as glycerol, DMSOl and formamide. Osmotic cryoprotectants increase intracellular osmotic pressure and reduce volume loss during freezing. thereby helping maintain cell viability. Particularly preferred penetrating cryoprotectants are: Pharmaceutical grade DMSO with a final concentration of 4-20%, preferably 7.5-10 % final concentration. At these concentrations (resulting in cell concentrations discussed below) The use of DMSO in a solution containing a total of about 0.002 to about 1 ml of DMSO This should result in cells being resuspended in the solution. As discussed in more detail below, DM This low total donor population of SO reduces the efficacy of therapeutically administered cells for transplantation. Improve yourself.

Membrane stabilizers stabilize cells by helping to reduce cell damage due to dehydration during freezing. It is thought that this helps maintain cell viability. Examples of membrane stabilizers include hydroxyethyl Contains chilled starch (HES), polyvinylpyrrolidone (PVP), and glucose. be caught. A particularly preferred membrane stabilizer is low molecular weight HES (Aoll[!rtca n　Crltical　Care.

McGaw Park, iI 1. ), 4 to 9%, preferably about 6 % final concentration.

- As shown in Figure 2, proteins can be dehydrated during freezing in the same way as membrane stabilizers. It is thought that it functions by reducing cell damage caused by suitable protein Examples of quality include albumin (animal or human), hemoglobin, animal serum (e.g. 1-IYcLONE@, Logan, Utah), and human plasma (Pug et 5ound Blood Center.

5eattle, Wastt or other blood center, blood bank, or plasma center Includes: Kumbaku substance can be used in a wide range of concentrations. It can be used with a solution width of about 5 to 9096 mm. However, protein If the solution is used with media containing Ca"+ or Mg" ions, protein 5-2 per ml of the solution to prevent coagulation or flocculation of cells due to It is preferred to include 0 units of heparin, preferably about 10 units of heparin. It should be noted that Particularly preferred proteins for use in the present invention are human This is autologous plasma.

This is because there is a possibility of contamination of the transplanted cells with foreign viruses (e.g., HIV). avoidance of risks that may occur when using potash-incompatible serum (e.g., fetal bovine serum). This is because it can overcome certain compatibility difficulties. In a preferred embodiment, the The plasma obtained from the donor was first centrifuged at 1.50×g to remove cell debris. to generate autologous plasma. Next, separate the supernatant liquid and add 10.000~+5. ,oo Centrifuge at oXg to remove particulates. Next, pass the clear liquid through a 0.8μ filter. The plasma is then passed through a 0.2μ filter to further clarify and sterilize it. Ru. In a preferred embodiment of the invention, autologous at a final concentration of about 10-20% of the solution. Use plasma.

In addition, nutritional supplements containing no or minimal amounts of protein fined supplements) in place of protein may be used to reduce cell damage due to dehydration during freezing. Particularly preferred regulations The nutritional supplement is ExVivo.

As indicated above, solutions preferably used in the present invention include media, proteins, and penetrating cryoprotectants. In one embodiment, the solution is about 20 Contains TCl 99 medium containing % autologous plasma and 7.5% DMSO.

The viability and recovery rate of cells for transplantation can be substantially maintained during freezing and thawing. It is also necessary to resuspend the cells in a sufficient volume of solution. In particular, cells at low concentrations Resuspension greatly reduces the number of viable cells recovered after freezing.

On the other hand, when cells are resuspended at high concentrations, they form clumps, thereby The number of cells that can be harvested is limited. Thus, for the present invention: 1. Approx. cells for transplantation 1 x 107 to 100XIO' cells/m summer concentration, preferably from about 10XIO'' to about , 10XIO' cells should be resuspended to a concentration of /''mI.

The resuspended cells are then subjected to a first set of conditions that substantially maintain cell viability. Freeze. Simply put, if you freeze cells too quickly, ice nucleation will occur. It begins to form ice crystals that can destroy the cells. Also, freeze the cells too slowly. This results in cellular dehydration. Accordingly, in one embodiment, the cells are Freeze at speed. This method is particularly preferred for solutions that do not contain HES. -4℃( At this temperature, cells emit heat called the “heat of fusion”) to maintain a controlled freezing rate. Since it is often difficult to maintain the Cryomed's Model l 010 (Cryom Speed control refrigeration systems such as ed, New Battimore, Mich. C may also be used. As shown in Figure 1, the refrigeration equipment intensively cools the temperature at approximately -4°C. As a result of cooling, a constant rate of temperature decrease is maintained in the cell suspension.

In particularly preferred embodiments, freezing comprises (a) cooling the cells to about 4°C; (b) Cool the 4°C cells at a rate of about 1.0°C/min until the cells reach about -4°C. (C) The -4°C cells are heated at about 0.5°C/min until the cells reach about -20°C. (d) cooling the -20°C cells at a temperature of about -40°C until the cells reach about -40°C; cooling at a rate of 1.0°C/min; and (e) cooling the -40°C cells until the cells are about - cooling at a rate of about 10°C/min until reaching 90°C.

However, it should be noted that other conditions may be used to freeze cells for transplantation. Should. For example, for cells resuspended in a solution containing about 6% HES Alternatively, they may be frozen by placing them directly in a -85°C freezer. Next, Tokoro If desired, they may be placed in liquid nitrogen.

Resuspended cells (A, cooled in any sterile Bayer suitable for storage in liquid nitrogen) Can be frozen. Particularly preferred is a Cryotube. s) (Corning Glass Works, Corni, ng, N ,Y,). Alternatively, if desired, cells for transplantation may be frozen directly in the syringe. good. Freezing purified cells for transplantation in a syringe allows for both freezing and administration to the patient. This is particularly advantageous since the same container can be used for both applications. This is due to possible contamination or Limit cell loss and improve the speed with which slightly previously thawed cells can be given to patients be able to. Also, Fenwacl or Delne The cells may be frozen in commercially available freezing bags from d).

As indicated above, freezing is followed by a second set of conditions that substantially maintain cell viability. Thaw cells under conditions. In a preferred embodiment, the cells are rapidly, preferably Thaw in a 37°C water bath.

Once the cells have been thawed, you can take steps to reduce the concentration of osmotic cryoprotectants or other excipients. They may also be diluted to return the cells to their normal state. for example, Cells equilibrated in a solution containing 10% DMSO have an osmotic pressure of 1800 mosm ( osmolarity), but physiological fluids have an osmolality of 300 mosm. . When thawed cells are immediately placed in a physiological solution, the cells quickly absorb water. is equal to the osmotic pressure of This rapid absorption of water causes many cells to lyse. follow It is especially preferred to slowly dilute the thawed cells with a physiological buffer such as PBS. Yes. This is because water and dissolved compounds have time to equilibrate across the cell membrane. 1, thereby limiting dangerous cell swelling. DMSO A particularly preferred method for reducing concentration is described in Example 6 below.

Administration of purified cells for transplantation Patients can become immunocompromised for a variety of reasons. For example, due to congenital genetic abnormalities , due to disease, or due to the use of toxic chemicals or radiation in the treatment of cancer. Patients can become immunocompromised. a therapeutic amount of cells for transplantation, and a total of about 0 , 002 to about 1 ml of an aqueous solution containing DMSO to Can be treated. In the context of the present invention, a “therapeutic amount” of cells for transplantation refers to This refers to the number of [CD34-positive] cells required to regenerate a person's immune response. requested The number of cells produced is about 0, +xio" to about 20XiO" cells/ml/g. . However, at least 0.75XIO' cells/ml/kg (patient weight) is particularly preferable. Thus, for a patient with body frlOO kg approximately 75X1.0' cells can be administered. The cells can be harvested from an IV bag or directly from a syringe. It should be injected into a vein by injection. Below is an example of this operation and the benefits obtained. This will be explained in detail in Example 11.

The following examples are given by way of illustration and are not intended to be limiting.

Preparation of avinonated biogel A, Carboxylation of polyacrylamide gel 17 g dry biogel (Bio gel) P-60 (trademark) (50-100 mesh (wet product), coarse beads) ( BIORAD, Catalog No.] 50.1630°Rie hmond, Calif, ) to 1,51 0.5M NaHCOslo, 5 Add to M NatcOs. Adjust the pH to 1O55 with NaOH and add to the beads. Be careful not to damage the mixer (RZR), Car4amo, Wi Stir for approximately 20-30 minutes with . The mixture is then placed in a 60°C water bath. The mixture reached a temperature of 60°C Afterwards, incubate for a further 2 hours (at 60° C.) with occasional stirring.

The mixture is then removed from the water bath and placed in a water bath to reduce the mixture temperature to room temperature.

After washing the beads several times with distilled or deionized water, remove the beads from a coarse glass connected to a vacuum source. Wash several times with PBS using a filter. The carboxylated gel was placed in PBS. Can be stored at 4°C (and stable for up to 1 year if sterilized or stored with a preservative). be.

B. Composite of carboxylated biogel with avidin. Filtered through a glass filter and measured the amount of PB from the carboxylated Iogel. Excluding S. The gel is then equilibrated in distilled or deionized water for 15-30 minutes. Ru. Upon equilibration in water, the gel expands to a volume approximately four times that measured previously.

For each 1 ml of the gel (first measured in PBS), add 10 ml of distilled water. Or resuspend in deionized water.

30 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( EDC-HCffi) (Jig 7-Chemical Co., Catalog No. E 7750. St. Louis.

Mo) is added to each 1 ml of gel measured first. Add HCf dropwise to adjust the pH. (Adjust to 5.5. Be careful to maintain pH at 5.5. If less than 5.0 or A pH higher than 6.0 significantly reduces the activity of the biogel. Add this mixture to 5 Stir for a minute.

Avinone (International Enzymes, Inc., Fallbrook, Ca. 1if,) is dissolved in deionized water at a concentration of 10-1.00 mg/ml. Next Add each iml of Img of avidin (value measured initially in PBS) to the gel. Add quickly. This mixture is mixed for 1.5 hours. Next, add 2M glycine to a final concentration of 0.2M glycine in the mixture and stir for an additional hour.

Washed several times with PBS using a coarse glass filter and vacuum and incubated in PBS at 4°C. Store in. The gel is stable for about I years.

Example 2 Isolation of cells for transplantation A, Isolation of pial cells. Centrifuge the bone marrow sample at 240Xg for 15 minutes. Remove the plasma (for later use) Centrifuge the remaining buffy coat cells again at 240Xg for 15 minutes. to remove red blood cells. R. buffy coat cells by centrifugation for 10 min at 180 PMIt'2DO wash. Next, the cells were treated with 1xl in t-RPMI + 1xBSA. Resuspend to a final concentration of O” leukocytes/m+.

B, Incubation of pial cell suspension with antibodies to a final concentration of 20μ g/ml biodinylated anti-CD34 antibody (CellPro@, Bothel 1. Incubate for 25 minutes at room temperature with Wash. Then 18 The antibody-cell mixture was washed twice with PBS by centrifuging for 10 min at 0Xg. Wash. The cells are then resuspended in PBS at a concentration of 1X10'' leukocytes/ml. Ru.

C. Column operations and results CEPRATE LC (trademark XCe11P) essentially in accordance with the manufacturer's instructions ro@.

Bothell, Wash, ) using a separation device! child. To explain briefly, the applicable Set up the equipment 7, connect the tubes, fill with reagents, and start operation with antibody-treated cells. did. Pump the cells through the column, wash the column with PBS, and then adsorb The cells were released using a magnetically driven stirring blade. The adsorbed cells were collected in a collection bag. .

D. Result ], 0XIO' bone marrow cells are passed through the column, and 200XIO@ cells are passed through the column. The cells were collected in a collection bag using binding (,,).The viability of the collected cells was It was 91% as determined by the Roux exclusion test. , these collected cells were subjected to FAC5 analysis. It was determined to be 75.9g CD34' by a standard method.

Example 3 Enrichment of cells for transplantation Gently invert the collection bag containing purified transplantable cells to mix the cells.

Cells were then transferred to two sterile 50 ml centrifuge tubes coated with autologous plasma. vinegar. Rinse the cell collection bag using 30 ml of TCI 99. 3 bottles of rinsing liquid Place in a separate 50ml tube. The bag was then washed with 20 tnl of TCI 99. Rinse for the 17th time, also into the third 50ml separation tube. Separation of the three Centrifuge the tube at 150Xg for 10 minutes.

Example 4 Remove the supernatant from the concentrated cells for transplantation. Two separation tubes with large pellets 1 ml of cell culture medium for transplantation (Te199.40% autologous plasma and IOU/ ml heparin) and mix into the rinse solution of the small pellet. This is 5 Approximately 2.25 ml of cells with a concentration of 0xlO' to 100xlO' cells/ml resulting in a suspension.

4. Add TCl 99 containing 10 U/ml heparin to the cell suspension. Bring to a final volume of 5 ml. Then 0.9 ml autologous plasma and 0.34 mt DM Add SO.

Add an equal volume of cryoprotective medium (Tel 99 containing 15% DMSO) to the cell suspension. Add gradually. Divide 4.5ml into each crya dupe and fry. Place it in the "pre-cooling chamber" of the speed control refrigeration system. Added the same volume of cryoprotective medium. A "dummy" tube is prepared and placed in the freezer. To record the freezing rate during extubation. For this purpose, a thermoelectric device χj is placed inside the extubation tube.

Example 5 Freezing cells for transplantation Cells for transplantation are frozen at a controlled rate from room temperature to -90°C as shown in Figure 1. . Briefly, this method consists of the following steps: (a) cooling the cells to approximately 4°C; (+)) The 4°C cells were heated at about 1.0°C/min until the cells reached about -4°C. (C) the -4°C cells until the cells reach approximately -20'C; (d) cooling the -20"C cells at a rate of about -4"C; Cool at a rate of approximately 1.0°C/min until reaching 0°C; and cool the -40'C cells. , and cooled at a rate of about 10.0°C/min until the cells reach about -90°C. In Figure 1 As shown, this experimental procedure results in a controlled rate of freezing.

Mugakudan Thawing cells for transplantation The cryotube containing the purified transplant cells was removed from the liquid nitrogen and placed in a sterile zipper bag. put it in the group. Place the bag containing the tubing in a 37°C water bath and agitate.

Once the last ice crystals have melted, transfer the contents to a 50ml centrifuge tube.

4.5 ml warmed transplantation cell dilution medium (TC199 and fOU/ml ~ ) into the 50 ml separation tube slowly (drop by drop at first). Addition of this medium should take approximately 2 minutes. After the first dilution, the final volume is 30ml. Cells for transplantation were purified and cryopreserved as described in Examples 2-6. Bone marrow? They also prepared buffy coats and treated them in the same way as the cells for transplantation, except that they were not purified.

As shown in Figure 2, purified cells for transplantation are significantly more viable than whole bone marrow. There were significantly more total cells (and CFCs) recovered.

Example 8 Measurement of CFC viability and recovery of Iscot supplemented with 2mML-glutamine and 50mg/ml gentamicin. Methylcellulose (Terry FOX Laboratories, Van Couver, Br1tish Columbia.

1 ml per 35 mm plate (Canada) was warmed to 37°C. correctness of measurement To improve accuracy, cells were diluted 3-fold and plated in triplicate. plate The highest number of cells was 3X! Except for purified cells plated at 03 and below. It was 10'/plate. Spread the cells evenly over the surface of each plate, then Incubate for 10-14 days in a humidified incubator at 37 °C in 5% CO2 air. Cubated. Count colonies containing more than 50 cells, CFU-GM, B Recorded as FU-E, or other (e.g. CFU-GMMM). various The number of colonies of each type was summed to give the total number of colony forming cells (CFC).

2.5X10', 5X10', 1oxto", 25X1O', 40X10', and Examples except that the cells were frozen at different concentrations including 50X IO' cells/ml. Cells for transplantation were purified and cryopreserved as described in Sections 2 to 6 above. Shown in Figure 3 When cells were frozen at 10XIO'~40XIO@cells/ml, Effect of DMSO on C-time cryopreservation As in Examples 2-6 except that cells were frozen at final concentrations of 7.5% and 10% DMSO. Cells for transplantation were purified and cryopreserved as described above. As shown in Figure 4, When using 7.5% DMSO, cell viability, recovery, and CFC recovery were It was significantly better.

Example 1.1 Administration of cells for transplantation Bone marrow was collected from 4 breast cancer patients by iliac crest aspiration. then kill tumor cells Therefore, the patient was treated with chemotherapy.

Cells for transplantation are purified from the bone marrow of the patient and administered to the patient essentially as described above. did. The results of this treatment are shown in Table I below.

59.8. 98 25 24 13 84.6 1.64 17 36 9 51 4, 32 i 3 16 3 57 1.05 32 30 22 Generally, engraftment (patient's granulocyte count is 500 cells/ It takes about 20 to 35 days (the number of days it takes to rise above lt1). Purified transfer In the above patients treated with transplanted cells, survival was demonstrated after only 13 to 32 days. . Furthermore, as shown above, the total volume of DMSO injected into the patient is is significantly different. In particular, about 0.002 to about 1 ml total DMSO (0.67 5 ml) was injected into each patient. Cell aggregates and hemoglobin are , were not detected in autologous transfused cells. Thus, none of these patients such as heart disease, headache, chills, dizziness, vomiting, arrhythmia, high blood pressure, difficulty breathing, or kidney failure. There were no toxic symptoms commonly associated with bone marrow autotransfusion.

Additionally, platelet recovery is often delayed in patients receiving autologous bone marrow transplants, and a significant number of blood cells are lost. Platelet transfusions (about 30 on average) are required. However, patients treated with the above each required (on average) only 12 units of platelets. this Decreased platelet usage is associated with blood-borne diseases. e) risk of transmission, alloimmunization, and cost burden (each unit of platelets costs approximately $300 to the patient); reduce the possibility of Thus, the present invention offers considerable benefits over conventional cryopreservation methods.

Although specific embodiments of the present invention have been described here for explanation, it is clear from the above that the present invention Understand that various changes can be made without departing from the idea and scope. There will be. Accordingly, the invention is not limited except as by the appended claims. .

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Claims (18)

[Claims] 1. (a) purifying cells for transplantation from a suitable blood product; (b) purifying the purified cells for transplantation; (c) enriching the enriched cells with substantial loss of cell viability during freezing and thawing. resuspend in a solution maintained at a concentration of approximately 10 x 10 to 40 x 10 cells/ml; and (d) a set of conditions that substantially maintain cell viability for said resuspended cells. A method of preparing cells for transplantation for future use, including freezing them under. 2. (a) purifying cells for transplantation from a suitable blood product; (b) purifying the purified cells for transplantation; (c) enriching the enriched cells with substantial loss of cell viability during freezing and thawing. a solution containing a total of 0.002 to about 1 ml of DMSO resuspend in; and (d) cooling the resuspended cells under a set of conditions that substantially maintain cell viability; A method of preparing cells for transplantation for future use, including freezing. 3. The freezing step is followed by thawing under a set of conditions that substantially maintain cell viability. 3. The method of claim 1 or 2, further comprising: 4. 4. The method of claim 3, wherein the cells are thawed in a 37<0>C water bath. 5. Following the thawing step, it is recommended to slowly dilute the thawed cells with physiological buffer. 4. The method of claim 3, comprising: 6. The purification process involves placing blood products on an immunoaffinity column that purifies cells for transplantation. 2. The method of claim 1, comprising passing. 7. 2. The method of claim 1, wherein the enriching step comprises centrifuging the cells for transplantation. 8. Concentrated cells for transplantation to a concentration of about 10 x 106 to about 40 x IO6 cells/ml. 3. The method of claim 2, further comprising resuspending. 9. 3. The method of claim 1 or 2, wherein the solution contains about 6% HES. 10. 5. The solution comprises a total of about 0.002 to about 1 ml of DMSO. The method described in 1. 11. Claim wherein the solution comprises a culture medium, a protein, and an osmotic cryoprotectant. The method described in 1 or 2. 12. The medium consists of RPMI1640, TC199, and Iscove DMEM. 12. The method of claim 11, wherein the method is selected from the group. 13. Claim 1 or 2, wherein the freezing step comprises freezing the cells at a controlled rate. Method described. 14. The freezing process at a controlled rate (a) cooling the cells to about 4°C; (b) the 4°C cells at a rate of about 1.0°C/min until the cells reach about -4°C; Cool; (c) The cells at -4°C are heated at a rate of about 0.5°C/min until the cells reach about -20°C. cooled at (d) The cells at -20°C are heated at a rate of about 1.0°C/min until the cells reach about -40°C. cooling at a rate; and (e) The cells at -40°C are heated at about 10.0°C/min until the cells reach about -90°C. 14. The method of claim 13, comprising cooling at a rate of . 15. (a) a therapeutic amount of cells for transplantation; and (b) a total of about 0.002 to about 1 ml of cells for transplantation; A composition comprising an aqueous solution containing DMSO. 16. 16. A method of treating an immunocompromised patient, the patient comprising: A method comprising administering a composition. 17. A syringe containing the composition of claim 15. 18. An infusion bag containing the composition according to claim 15.