WO2006011681A1

WO2006011681A1 - Preservation method and transport method for blood for leucocyte incubation, preservation method and transport method for peripheral blood monocytes and method of incubating leucocytes using the same

Info

Publication number: WO2006011681A1
Application number: PCT/JP2005/014364
Authority: WO
Inventors: Hidemasa Jinguji; Atsutaka Noguchi; Shiho Sato; Kazutoshi Sato
Original assignee: Medinet Co., Ltd
Priority date: 2004-07-30
Filing date: 2005-07-29
Publication date: 2006-02-02
Also published as: JPWO2006011681A1

Abstract

It is intended to provide a method whereby leucocytes can be easily separated from the blood collected from a patient and preserved and/or transported while sustaining a high proliferation ability, thereby achieving excellent effects as preservation and/or transport methods for the blood for leucocyte incubation to be used in immunological cell therapy. It is also intended to provide an incubation method with the use of leucocytes preserved and/or transported by the above methods. Namely, the blood for leucocyte incubation is preserved at 16 to 22oC so that peripheral blood monocytes can be well separated 10 hours, in particular, 30 hours after the blood collection and a high proliferation ability can be sustained. In the case where peripheral blood monocytes are separated before the preservation, the temperature range suitable for the preservation and/or transport is from 5 to 22oC.

Description

Leukocyte culture blood storage method, transport method, peripheral blood mononuclear cell storage method, transport method and leukocyte culture method using them Technical Field

The present invention relates to a method for preserving blood for leukocyte culture, a method for transporting blood for leukocyte culture, a method for preserving peripheral blood mononuclear cells, and a method for transporting peripheral blood mononuclear cells. Furthermore, the present invention relates to a method for culturing leukocytes using them. Background art

The most common cause of death among Japanese is malignant neoplasm (hereinafter referred to as cancer). There are three major therapies for cancer, surgical treatment, chemotherapy, and radiation therapy, but there are problems such as difficulty in treatment and side effects.

In recent years, in addition to the above three major therapies, immune cell therapy has been carried out as a new treatment for cancer, and this immune cell therapy has few problems such as treatment difficulties and side effects like the above three major therapies. Because of it. Immune cell therapy is also attracting attention as a new treatment for infections, especially viral infections.

Here, immune cell therapy refers to collecting blood from a patient, separating and culturing leukocytes contained in the blood, activating and Z or proliferating the leukocytes, and activating and / or proliferating the leukocytes. This refers to a therapy in which leukocytes (including those prepared by using activated and / or expanded leukocytes as an instillation) are returned to the patient's body. For example, activated autolymphocyte therapy (including activated lymphocytes including L AK cells (Lymphophocine Acivated K i 1 1 er cells)), dendritic cell vaccines, CTL therapy (Cytoto Xic TL ymphocyte) Therapy).

In order to carry out this immune cell therapy, it is necessary to have cell culture facilities that cultivate leukocytes, but medical institutions (hereinafter referred to as hospitals) throughout the country do not have such facilities. Therefore, in order to facilitate immune cell therapy even in hospitals that do not have cell culture facilities, blood for leukocyte culture is transported from hospitals to cell culture facilities, and after leukocyte culture, It must be prepared and transported from a cell culture facility to a hospital.

Even without transport, depending on the schedule for returning to the patient's body, it may be necessary to store the leukocyte culture blood collected from the patient in advance for a certain period of time. In that case, it was necessary to maintain the separation and culture growth of leukocytes from the blood for culturing leukocytes after storage and / or transportation.

However, in general, for blood storage and Z or transport, only the storage temperature of whole blood, red blood cells or platelets used for transfusion is known. Disclosure of the invention

The present invention has been made in view of the above circumstances, and provides a method for easily separating leukocytes from blood collected from a patient and storing and transporting or transporting them while maintaining high proliferation ability. The purpose is to provide. Furthermore, it aims at providing the culture method using the said white blood cell.

According to the present invention, by storing the blood for leukocyte culture at 16 to 22 ° C., it is possible to separate peripheral blood mononuclear cells after blood collection for 10 hours or more, especially 30 hours or more. It is possible to maintain a high proliferation ability. In order to maintain the above temperature, it may be maintained with a normal thermostatic device, and when transported, it may be transported in a state of being placed in the thermostatic device. As a simple method, the cold storage for maintaining the temperature of the present invention may be stored and Z or transported in a normally used cold storage box having excellent heat insulation. In addition, blood collected from a patient may be subjected to an operation such as centrifugation in advance before storage to separate peripheral blood mononuclear cells.

Leukocytes in the present invention mean granulocytes such as eosinophils, basophils, and neutrophils; monocytes; lymphocytes such as T lymphocytes, B lymphocytes, and natural killer cells. The leukocytes in the blood for culturing leukocytes preserved in this state maintain excellent separation and proliferation ability, including lymphocytes in immune cell therapy such as activated autolymphocyte therapy, dendritic cell vaccine, and CTL therapy. It is useful for the culture of leukocytes. Brief Description of Drawings'

Figure 1 (a) shows the effect on the number of recovered cells at storage temperatures of 2 ° C, 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25, and Figure 1 (b) shows peripheral blood. Indicates the number of red blood cells mixed in the mononuclear cell fraction.

Figure 2 shows the effect on cell growth rates at 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25 ° C at storage temperature 2. The vertical axis shows the magnification when the number of cells before culture is 1.

Figure 3 shows the effect on the number of recovered cells at storage temperatures of 16 and 18 ° (: 20 ° C, 22 ° C).

Figure 4 shows the effect on cell growth rate at storage temperatures of 16, 18 ° C, 20 ° C, and 22 ° C. The vertical axis shows the magnification when the number of cells before culture is 1.

Figure 5 shows a storage temperature of 5 ° C for samples from which peripheral blood mononuclear cells were separated before storage. The number of recovered cells at 10 ° C, 15 ° C and 20 ° C is shown.

Figure 6 shows the cell growth rate at storage temperatures of 5 ° C, 10 ° C, 15 and 20 ° C for samples subjected to peripheral blood mononuclear cell separation before storage. The vertical axis shows the magnification when the number of cells before culture is 1. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described.

Collect blood from patients with cancer and / or infection. The collection method is not particularly limited, but a commonly used blood collection tube may be used.

Here, peripheral blood mononuclear cells may be separated. In general, any method for separating nucleated cells from erythrocytes can be used as a method for separating peripheral blood mononuclear cells. For example, a method using a Ficoll pack (Ficol 1 -Paque) density gradient is generally used.

By separating peripheral blood mononuclear cells, the storage temperature range described above is expanded to 5 to 22 ° C. The isolated peripheral blood mononuclear cells may be suspended in any carrier as long as they are isotonic with cells, but are preferably suspended in autologous plasma.

Next, place the blood collection tube containing blood together with a cold insulation so that 16-22t: can be secured in the cold storage box, and store and transport it.

After that, blood is separated (this operation is not necessary if peripheral blood mononuclear cells have been separated in advance), and lymphocytes are cultured. The culture method is not particularly limited, and any culture method that is generally used in the field may be used, and when used for activated autolymphocyte therapy, a method using an anti-CD3 antibody and IL-12 is particularly preferable.

The anti-CD3 antibody may be added to the medium or immobilized on the culture vessel, but it is preferable to seed the lymphocytes on a culture vessel such as a flask in which the anti-D3 antibody is immobilized. It is preferable to add IL-12 so that the concentration in the medium is 100 to 2000 IU / mL.

Culture, 34~38Τλ preferably at 37, 2-10%, preferably carried out at 5% of C0 ₂ conditions, the incubation period is from 1 day to 20 days, it is not preferable particularly about 1-2 weeks.

The medium that can be used is not particularly limited, but AIM-V medium (Invitrogen), RPM 1-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen), Iskov medium (Invitrogen), KBM medium (Kohjin Bio), Commercially available media used for cell culture such as ALyS medium (Laboratory for Cell Science) can be used. If necessary, 5-20% bovine serum, fetal bovine serum, human serum, human plasma, etc. can be added.

The culture vessel is not particularly limited, and is usually for culture used in the field. Plates, petri dishes, flasks, bags, etc. can be used. The concentration for seeding each cell group can be freely set according to the situation. The lymphocytes harvested after separation and culture from the collected blood are prepared as injections using commonly used carriers. The carrier to be used is not particularly limited. For example, when it is prepared as an instillation agent, an isotonic solution such as physiological saline or PBS (phosphate buffered physiological saline) can be used. A serum component such as albumin may be added to the isotonic solution.

Next, inject the infusion, such as a drop, together with the refrigeration agent so that the temperature can be kept at 0 to 6 ° C in the refrigeration box, and store and transport it or transport it.

The lymphocytes preserved in this state maintain a high viable cell rate and an IFN-a-producing cell rate, and are useful as an injection for immune cell therapy. In particular, it is useful as an injection for use in immune cell therapy for cancer and Z or infection.

In the present invention, as described above, by storing and / or transporting the collected blood at 16 to 22 ° C., the separation of peripheral blood mononuclear cells from the collected blood is good and the proliferation is high. Can be maintained in a state. On the other hand, when peripheral blood mononuclear cells are separated from the collected blood in advance, the storage temperature range is expanded to 5 to 22 ° C while maintaining a high proliferative state.

Using such peripheral blood mononuclear cells maintained in a highly proliferative state, lymphocytes obtained by culturing are stored and transported or transported at 0-6 ° C. This makes it possible to obtain lymphocytes that maintain a high viable cell rate and an IFN-producing cell rate while maintaining a high proliferative state. Therefore, in lymphocyte culture, there is an excellent effect by imposing different treatments on the sample blood before the culture and the lymphocytes obtained after the culture, and a more excellent effect can be obtained by combining these treatments.

The culture method used here may be cultured not only under the above-mentioned conditions but also under conditions generally known in lymphocyte culture.

As described above, the case of lymphocyte LAK cell culture has been described in detail. However, other leukocytes may be cultured by a method generally known in the art. Example

Hereinafter, the present invention will be described in detail with reference to examples. However, it goes without saying that the present invention is not limited to this. Example 1

Transfer 7.5 ml of peripheral blood from a healthy donor to a vacuum blood collection tube (vacutina CPT: Blood was collected by Kuton (Dickinson).

The blood that entered the vacuum blood collection tube was stored at each temperature for 30 hours.

Thereafter, the cells were left at room temperature for 1 hour, and baboon peripheral blood mononuclear cells were separated by centrifugation (1710Xg, 20 minutes).

The number of collected cells was measured using a flow cytometer (Cytomics FC500: Beckman Cole Yuichi).

Using the above peripheral blood mononuclear cells in an anti-CD3 antibody (Orthoclone: Janssen Pharma) in a solid-phase flask (Sumitomo Beklite) using a medium (KBM400: Kojinbayo), IL-2 is 280 I UZmL added to a cultured (37 ° C, C_〇 _2: 5%) was performed.

The number of LAK cells after 7 days of culture was measured with a hemocytometer.

1) Effect of storage temperature 2, 6 ° C, 10 ° C, 14 ° C, 18 ° C, 25 ° C

Figure la shows the number of T cells collected in peripheral blood mononuclear cell fractions from peripheral blood stored at 2, 6 ° C, 10 ° C, 14, 18 and 25 ° C for 30 hours. b shows the number of red blood cells mixed in the peripheral blood mononuclear cell fraction.

At storage temperatures of 2, 6 ° C, and 25 ° C, the number of recovered T cells was large, but the contamination rate of erythrocytes was high. It was found that storage at about 10 ° C to 18 ° C is suitable for recovering T cells with little red blood cell contamination.

Figure 2 shows LAK cells grown from peripheral blood mononuclear cells collected from peripheral blood stored at storage temperatures of 2 ° C, 6 ° C, 10 ° C, 14 ° C, 18 ° C, and 25 ° C for 30 hours. The growth rate was shown.

After 7 days of culture, the storage temperature of LAK cells was 50 times at 18 ° C, 34 times at 14 ° C, and 20 times at others. When inducing LAK cells using stored blood, it was found that a storage temperature of around 18 is suitable.

2) Effect of storage temperature 16, 18 ° C, 20t, 22 ° C

Fig. 3 shows the number of T cells recovered from the peripheral blood stored for 30 hours at storage temperatures of 16 ° C, 18 ° C, 20, and 22 in the peripheral blood mononuclear cell fraction.

It was revealed that the number of recovered cells was high at a storage temperature of 20 ° C, and that there were few erythrocyte contamination under each condition.

Figure 4 shows the proliferation rate of LA cells proliferated from peripheral blood mononuclear cells collected from peripheral blood stored at storage temperatures of 16 ° C, 18 ° C, 20 and 22 ° C for 30 hours.

It was revealed that there was no significant difference in the growth rate of LAK cells after 7 days of culture under any condition.

In this way, when considering the separation and recovery of peripheral blood mononuclear cells and the proliferation rate of LAK cells, in the case of inducing LAK cells using stored blood, in the temperature range of 16 ° C to 22 ° C It has been found that storage of blood for lymphocyte culture is most effective. Example 2

Effect of storage temperature on the number of recovered cells and cell growth rate when peripheral blood mononuclear cells are separated before storage>

7.5 ml of peripheral blood was collected from a healthy donor into a vacuum blood collection tube (Baccutina CPT: Becton 'Dickinson').

Thereafter, human peripheral blood mononuclear cells were separated by centrifugation (1710 Xg, 20 mn.).

Peripheral blood mononuclear cells were stored in autologous plasma for 30 hours at each temperature. The number of collected cells was measured using a flow cytometer (Cytomics FC 500: Beckman Cole).

Using the above peripheral blood mononuclear cells in an anti-CD3 antibody (Orthoclone: Janssen Pharma) in a solid-phase flask (Sumitomo Beichikrite) using a medium (KBM400: Kojin Bio), IL-2 is 280 IU / was added to a mL culture _{(37 ° C, C0 2:} 5%) was.

The number of cells (LAK cells) after 8 days of culture was measured with a hemocytometer.

Figure 5 shows the number of sputum cells recovered from samples that were isolated from peripheral blood mononuclear cells before storage and stored at storage temperatures of 5 ° C, 10 ° C, 15 ° C, and 2 ° C for 30 hours.

It was revealed that the number of collected sputum cells in samples that had been subjected to peripheral blood mononuclear cell separation before storage was 5 to 20 and the dependency on storage temperature was low.

Fig. 6 shows the proliferation rate of LAK cells grown from samples that were isolated from peripheral blood mononuclear cells before storage and stored at storage temperatures of 5, 10 ° C, 15, and 20 ° C for 30 hours. LAK cell proliferation in samples from which peripheral blood mononuclear cells were isolated before storage was found to be 5 to 20 ° C, and the dependence on storage temperature was low.

Thus, it was revealed that when peripheral blood mononuclear cells were separated prior to storage, the temperature range suitable for storage expanded to 5-22 ° C. Industrial applicability

As described above, the method of the present invention provides a method for easily separating leukocytes from blood and preserving and / or transporting them while maintaining high proliferation ability. It has an excellent effect as a method for storing and / or transporting blood for lymphocyte culture in therapy. In addition, high proliferation can be expected by culturing leukocytes isolated from these lymphocyte culture blood.

Claims

The scope of the claims

I. A method for preserving leukocyte culture blood, wherein the leukocyte culture blood is stored at 16 to 22 ° C.

2. The method for preserving blood for leukocyte culture according to claim 1, wherein the blood for leukocyte culture is blood derived from a cancer and / or infectious disease patient.

3. The method for preserving leukocyte culture blood according to claim 1 or 2, wherein the leukocyte culture blood is lymphocyte culture blood.

4. A method for transporting blood for leukocyte culture, comprising transporting blood for leukocyte culture at 16 to 22 ° C.

5. The method for transporting leukocyte culture blood according to claim 4, wherein the leukocyte culture blood is blood derived from cancer and Z or an infectious disease patient.

6. The method for transporting blood for leukocyte culture according to claim 4 or 5, wherein the blood for leukocyte culture is blood for lymphocyte culture.

7. Peripheral blood mononuclear cells are isolated from leukocyte culture blood, and the isolated peripheral blood mononuclear cells are stored at 5 to 22 ° C. Method.

8. The method for preserving peripheral blood mononuclear cells for leukocyte culture according to claim 7, wherein the leukocyte culture blood is blood derived from a cancer and / or infectious disease patient.

9. The method for preserving peripheral blood mononuclear cells for leukocyte culture according to claim 7 or 8, wherein the blood for leukocyte culture is blood for lymphocyte culture.

Transport of peripheral blood mononuclear cells for leukocyte culture, characterized by separating peripheral blood mononuclear cells from leukocyte culture blood and transporting the separated peripheral blood mononuclear cells at 5 to 22 ° C. Method.

The method for transporting peripheral blood mononuclear cells for leukocyte culture according to claim 10, wherein the leukocyte culture blood is blood derived from a cancer and / or infectious disease patient.

12. The method for transporting peripheral blood mononuclear cells for leukocyte culture according to claim 10 or 11, wherein the leukocyte culture blood is lymphocyte culture blood.

1 3. In leukocyte culture, is the peripheral blood mononuclear cell used isolated from leukocyte culture blood stored and / or transported at 16 to 22 ° C? Alternatively, the method for culturing leukocytes, wherein the peripheral blood mononuclear cells are separated and transferred or Z or transported in 5 to 2 2 after separation of peripheral blood mononuclear cells from blood for leukocyte culture.

1 4. In leukocyte culture, peripheral blood mononuclear cells separated from leukocyte culture blood stored or transported in 16 to 2 2 or peripheral blood mononuclear cells were isolated from leukocyte culture blood in advance. After 5 to 2 2 stored and transported Z or transported peripheral blood mononuclear cells to anti-CD3 antibody and 1 0 0 to 2 0 0 0 1 UZmL IL-1 Presence, the process of leukocyte culture according to claim 13, wherein that you cultured 20 days in C0 ₂ under conditions of 2-10% in 34 to 38 ° C.

15. The lymphocyte culture is performed using the peripheral blood mononuclear cells, and then the obtained lymphocytes are stored and Z or transported at 0 to 6 according to claim 13 or claim 14. Leukocyte culture method. '.

16. The leukocyte culture method according to any one of claims 13 to 15, wherein the blood for leukocyte culture is blood derived from cancer and Z or an infectious disease patient.

17. The leukocyte culture method according to any one of claims 13 to 16, wherein the leukocytes are lymphocytes.