CN104204195B - Using memory T cell as the manufacture method of the lymphocyte populations of main component - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
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- A61P35/00—Antineoplastic agents
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Abstract
In lymphocyte from peripheral blood, T cells account for therein about 50% before activation culture is implemented, but the ratio will be reduced by activation culture.On the other hand, the occupation rate before culture starts for the memory T cell below half is increased by activation culture, is even up to 95%, but if continuing to culture, it will be gradually decreased.The problem of the present invention be easy in the way of without using antibody etc. and promptly a large amount of methods for preparing the positive memory T cells groups of CD45RO are provided, with the memory T cell group that is made by methods described for medicinal composition of main component etc..It is a feature of the present invention that manufacturing the substantial amounts of lymphocyte populations by main component of memory T cell by following process, the process is：Activation culture is carried out to the lymphocyte of collection, desired value has been reached using the occupation rate that the cell density of the lymphocyte populations in culture is more than certain threshold value as index, the memory T cell detected in lymphocyte populations, the cell mass is harvested, freezen protective is carried out；The Cell Cryopreservation is thawed, cultivated so that it is bred.
Description
Technical field
Drenched the present invention relates to a large amount of manufacture methods using memory T cell as the lymphocyte populations of main component, containing described
Manufacture system (combinations of the means such as device) of the medical composition of bar cell mass and the lymphocyte populations etc. is described big
Amount manufacture method is for easily largely obtaining the T that the CD45RO from the peripheral blood containing T cell is positive and CD62L is positive
The method for the T cell group that cell mass, CD45RO are positive and CD62L is negative.
Background technology
Lymphocyte receives much concern in recent years as the important means for supporting the immune system for body defenses.Lymph is thin
The surface expression of born of the same parents has the antigenic type referred to as differentiation antigen, and lymphocyte is classified as various Asias according to the antigenic type
Group (subset).For example, to whetheing there is CD3, (CD is differentiation cluster (cluster of according to the reactivity of monoclonal antibody
Differentiation abbreviation)) express and test, thus lymphocyte is distinguished as to the T lymph related to cellular immunity
(CD3 is positive, also is indicated as CD3+ for cell；Also the positive is recited as sometimes below+, feminine gender be recited as -) and with humoral immunity phase
Bone-marrow-derived lymphocyte of pass etc. (CD3 is negative, also is indicated as CD3-).And the respective function of these lymphocytes is also different.Further
Ground, the T lymphocyte (also referred to as T cell) related to cellular immunity is classified as express CD4 CD4 positive cells and expression
CD8 CD8 positive cells.In the past, think that CD4 positive cells play miscellaneous function, CD8 positive cells and play Carbazole alkaloid always
Sexual function, therefore so far, in most cases, will again be studied after CD4 positive cells and the separation of CD8 positive cells.
It is difficult that difference is carried out to lymphocyte by form, in most cases as above according to antigenic type will drench
Bar cell is divided into each subgroup and function is analyzed.But, it is to use the master corresponding with the antigenic type of each cell mostly
Will for monoclonal antibody (such as anti-CD4monoclonal antibody, anti-CD8 monoclonal antibodies) come what is carried out.Moreover, being to make lymphocyte
Reacted with combining the antibody on magnetic micro-beads grain (magnetic microbeads), utilize attaching from external container
The magnetic collection magnetic micro-beads of magnetite, separation are via cell of the antibody binding on the bead (beads) or using separating
Post is come the method that is separated.In addition, in order to largely obtain CD4 positive cells, CD8 positive cells, also having and utilizing microballon grain
Method by its it is individually separated after, further activated, the method for amplification cultivation (referring to patent document 2).In addition, recently, by
In the progress of determining instrument, lymphocyte is dyed using the fluorescent labeled antibody for respective surface antigen, used
The method that cell sorter (cell sorter) is differentiated is also carried out.In this method, in advance with fluorescent dye to for thin
The antibody of cellular surface antigen is marked, and labelled antibody is combined on lymphocyte, and the lymphocyte is supplied to using thin
The measure that born of the same parents' sorter is carried out separates so as to examine the expression status of surface antigen to subgroup.But, this method is deposited
(referring to non-patent literature 2) the problem of being only applicable on a small scale.Which kind of, no matter using conventional method, it can not all avoid with high price
Antibody numerous and diverse operation such as lymphocyte is marked, to obtain substantial amounts of cell need it is expensive laborious.
It is initially the report carried out by Sekine et al. on the manufacture method of activated T lymphocytes.This is to selectivity
Ground stimulates the propagation of activated T lymphocytes group so as to the report first for the method for obtaining the activated T lymphocytes group through propagation, tool
For body, wherein by human peripheral lymphocyte (PBMC：Peripheral Blood Mononuclear Cell) in immobilization
Carry out first phase culture in the culture flask of OKT3 antibody (it is one of anti-cd 3 antibodies), by the cell cultivated through this not
Second phase culture is carried out in the case of there is OKT3 antibody.The activated lymphocyte group obtained using this method further across
Culture in the presence of interleukin 2 (IL-2), can as cancer adoptive immunotherapy (adoptive
Immunotherapy) bestow to the patient that source is gathered as above-mentioned peripheral blood (referring to patent document 1).In addition, with same
Mode, the PBLC from donor is carried out in immobilization in the culture vessel of OKT3 antibody in the presence of IL-2
Culture, CD4 positive cells are separated from the activated lymphocyte group obtained, can be using as with for total cell 90%
More than amount contain isolated CD4 positive cells it is preparation, for prevent and treat infectious disease, tumor recurrence, itself
The medical composition of immunity disease is bestowed to patient (referring to patent document 2).As their lymphocyte source, it can be used swollen
Knurl lymphocyte infiltration (TIL：Tumor Infiltrating Lymphocyte), Cord blood, marrow etc. is (referring to patent document
2)。
Consider in addition from functionality viewpoint, sometimes by Lymphocyte subset be initial lymphocyte (
) and memory lymphocyte (memory lymphocyte) lymphocyte.Initial lymphocyte is not yet by antigenic stimulus
, cell surface expression have the lymphocytes of CD45RA antigens, it is known that its in regional nodes etc. met with antigen and by
To being activated during stimulation.Memory lymphocyte is to be subjected to antigenic stimulus (either differential stimulus is also non-spy
The opposite sex is stimulated) and express the lymphocyte for having CD45RO antigens.T lymphocytes and bone-marrow-derived lymphocyte can also be according to respective shapes
State is divided into T cells and memory T cell, naive B cell and memory B cell.Memory T cell can be further divided into effect type
Remember (effector memory；EM) T cell and central type memory (central memory；CM) T cell.Central type memory T
Cell, which has, goes back to the nest to the function of lymph node, the foreign matter and antigen met head-on in intrusive body, and effect type memory T cell has in this
The effect at position, seizure foreign matter and antigen where answering.Therefore, it is contemplated that contain preparation of the memory T cell group as main component
High therapeutic effect can be obtained in adoptive immunotherapy.Therefore, Osumi et al. is in order to be illustrated in adoptive immunotherapy
The mechanism of action of the middle activated lymphocyte used, is conceived to memory T cell subgroup, and the activation that activation amplification cultivation is obtained is drenched
The function of bar cell mass is analyzed.As a result, confirming that the effect type memory T cell included in activated lymphocyte group exists
Co-cultivation with tumour cell shows the cell inhibitory activity to cancer cell after starting 24 hours；On the other hand, central type
Memory T cell is showed to cancer cell after beginning and cancer cell are co-cultured, identified 48 hours of tumour antigen
Cell inhibitory activity, effect type memory T cell has been divided at that time (referring to non-patent literature 2).
Report on gathering memory T cell, can trace back to the document delivered by Sekine et al. in 2000 (referring to special
Sharp document 3), the lymphocyte from Cord blood is carried out activation culture in the presence of anti-cd 3 antibodies and IL-2, it is lived by it
Change propagation 7 days, the ratio that results acquisition has arrived CD3 positive cells is 98% and the ratio of CD45RO positive cells is 73%
Cell mass.But, the cell in this period, which exists, breeds the problem of insufficient, cell number is few.
Above-mentioned activated lymphocyte needs to bestow patient rapidly after Multiplying culture, it is difficult to carry out adapting to applying rapidly for the state of an illness
The qualitative control given and stablized.Therefore, it has been reported that in view of the convenience of patient, when preparing lymphocyte by patient's blood, in work
Change lymphocyte freezen protective after culture (referring to non-patent literature 1).Wherein calculate lymph that is chilled, thawing
Cell again in activated lymphocyte obtained from activation culture CD4 lymphocytes and CD8 lymphocytes occupation rate, conclusion
For：Nearly all population (population) is bred in an essentially parallel manner.In non-patent literature 3, disclose separation and come from navel
Activation culture is carried out after lymphocyte with blood and peripheral blood, afterwards freezen protective, defrosting for the time being, now to the Asia during culture
The result that group's change is tracked.Report, the lymphocyte from Cord blood and peripheral blood has following situations：It is used as note
The central type memory T cell for recalling the subgroup of T cell is bred rapidly after culture starts, in the height that propagation is reached in 6 days to 7 days
Peak, reduce thereafter, effect type memory T cell after incubation breed by the phase.In addition, Sekine et al. discloses following bestow with system
The manufacture method of agent：The lymphocyte of patient is set to carry out Proliferative Activated culture in vitro, then in ultra low temperature freezer
(freezer) interior freezen protective, adds physiological saline and is suspended immediately after being thawed when using by freezing state, thus be made to
Pharmaceutical formulation (referring to patent document 4).
As described above, being implied in adoptive immunotherapy using the validity of memory T cell, it may be said that great Liang Jian
Just it is social demand to gather the abundant component of memory T cell.But, present situation is not not to lymphocyte supplier
Force a large amount of easily collections in the case of very big burden for be supplied in clinic and needed for memory T cell method.
Prior art literature
Patent document
Patent document 1：Japanese Unexamined Patent Publication 3-80076 publications
Patent document 2：No. 4389039 publications of Japanese Patent No.
Patent document 3：Japanese Unexamined Patent Publication 2002-171966 publications
Patent document 4：No. 4395644 publications of Japanese Patent No.
Non-patent literature
Non-patent literature 1：Sekine T.,Human Cell,5(3)243-245(1992)
Non-patent literature 2：32nd cancer immunity surgery research association agenda (Program) the summary collection Heisei 23 of page 103
April in year
Non-patent literature 3：Shikamura M. et al., Biotherapy 23 (3) 257-262 (2009)
The content of the invention
In lymphocyte from peripheral blood, T cells account for therein about 50%, but the ratio before activation culture is implemented
Rate is reduced by activation culture.On the other hand, activation is passed through before culture starts for the occupation rate of the memory T cell below half
Cultivate and increase, be even up to 95%, but if continuing to culture, it will be gradually decreased.The problem of the present invention is to carry
It is provided with the mode without using antibody etc., prepares the positive memories of CD45RO in large quantities using easy and rapid high proliferation cultivation
T cell group method there is provided with by methods described make with memory T cell group for main component medicinal composition
Deng.
The present inventor etc. are for the purpose of improving the ratio of the memory T cell included in activated lymphocyte, various
Under conditions of research be repeated, work hard, as a result find：If by PBLC in anti-cd 3 antibodies immobilization
Activation culture is carried out in flask, in the nutrient solution containing IL-2, then over time, cell surface antigen is CD45RO
Positive and CD62L positive (CD45RO+, CD62L+) T cell group and CD45RO is positive and CD62L it is negative (CD45RO+,
CD62L- T cell group (i.e. memory T cell group) increase), the ratio occupied in lymphocyte populations rises.But further acknowledge
Arrive：Because total cell number is still less at this point in time, so also needing to continue to cultivate, but if continuing if cultivating, memory T
The ratio of cell mass switchs to reduce.Therefore, the present inventor etc. in the way of not making the ratio reduction of memory T cell group to making lymph
Cell is bred, manufactures studied with keen determination for the method for the cell mass of main component with memory T cell group in large quantities.It is tied
Really, in order to obtain memory T cell in large quantities, when continuation is cultivated up to the surface antigen in the lymphocyte populations in activation culture is
CD45RO+, CD62L+ and/or CD45RO+, the ratio of CD62L- T cell group rise to desired ratio and then harvest rising
To the desired ratio lymphocyte populations when, using cell density be more than certain threshold value as index, by the cell of the harvest
Freezen protective is carried out, activation amplification cultivation is then carried out again, above mentioned problem is thus solved.
That is, because it is found that rising and the cell density of activation culture lymphocyte populations of the occupation rate of memory T cell group
Between relation, therefore cell density to reach and more than certain threshold value as the index of harvesting mean that above-mentioned memory
T cell group has reached desired occupation rate.Thus, using easy and low cost the method as cell density that determines, with regard to energy
Enough detect the period for being most suitable for cell mass of the harvest with enough cell numbers and high memory T cell group's occupation rate.This
Outside, it was found that cell density during by inoculation is managed, can to the culture untill harvest during most closed
Suitable control is thin with enough cell numbers and high memory T cell group's occupation rate so as to harvest in a short time
Born of the same parents group.As described above, successfully developing easy in the way of without using antibody etc. and largely manufacturing at low cost with memory T cell
For the method for the lymphocyte populations of main component.
That is, the present invention includes the manufacture method of following [1]~[4]：
[1] using memory T cell as the manufacture method of the lymphocyte populations of main component, the manufacture method is utilized and wrapped successively
Following process (a)~(c) high proliferation cultivation is included, the process is：
Process (a), activation culture is carried out to the lymphocyte of collection；
Process (b), is risen based on lymphocyte Density Detection to the occupation rate of memory T cell group, by the lymph of process (a)
Cell carries out freezen protective；
Process (c), is thawed, activation culture to the lymphocyte being frozen；
[2] manufacture method as described above described in [1], it is characterised in that based on lymphocyte density to memory in process (b)
The occupation rate of T cell group rises the detection carried out using the lymphocyte number that is suspended in nutrient solution as index；
[3] manufacture method as described above described in [1] or [2], it is characterised in that the lymphocyte density in process (b) is：
The lymphocyte number being suspended in nutrient solution is 4 × 105Individual cell/more than mL；
[4] manufacture method as described above any one of [1]~[3], it is characterised in that activation culture be in IL-2 and
Cultivated in the presence of immobilization anti-cd 3 antibodies.
In addition, the present invention includes the manufacture method of following [5]~[9]：
[5] manufacture method as described above any one of [1]~[4], it is characterised in that process (c) was in 2 weeks after defrosting
Within complete；
[6] manufacture method as described above any one of [1]~[5], it is characterised in that using memory T cell be main
The lymphocyte populations of composition are that the occupation rate of memory T cell in lymphocyte populations is more than 75% lymphocyte populations；
[7] manufacture method as described above any one of [1]~[6], it is characterised in that memory T cell is to account for lymph
More than 70% ratio of cell mass contains central type memory T cell；
[8] manufacture method as described above any one of [1]~[7], it is characterised in that separated lymphocyte comes
From human peripheral；
[9] manufacture method as described above any one of [1]~[8], it is characterised in that lymphocyte populations are to be used to apply
Give to the cell mass of the third party.
In addition, the present invention includes following [10]~[13]：
[10] a kind of lymphocyte populations, it is characterised in that contain more than 75% memory T cell for accounting for lymphocyte populations；
[11] lymphocyte populations as described above described in [10], it is characterised in that containing accounting for more than the 70% of lymphocyte populations
Central type memory T cell；
[12] a kind of medical composition, lymphocyte populations described in the medical composition using above-mentioned [10] or [11] is have
Imitate composition；
[13] using memory T cell as the manufacture system of the lymphocyte populations of main component, the manufacture system for possess with
Under (A)~(D) manufacture system means, using high proliferation cultivation, means (A)~(D) be：
Means (A), the activation culture means of lymphocyte；
Means (B), the means that the occupation rate based on lymphocyte Density Detection memory T cell group rises；
Means (C), the freezen protective means of lymphocyte；
Means (D), the means thawed to the lymphocyte being frozen；
In accordance with the invention it is possible to easy and provide to contain in the activated lymphocyte of peripheral blood in large quantities
CD45RO+ and CD62L+, CD45RO+ and CD62L- memory T cell group are the lymphocyte populations of main component.Drenched to activation
When the propagation of the memory T cell group included in bar cell mass is tracked, the cell carried out in conventional method can not be used
The such numerous and diverse method using high price antibody of surface antigen analysis, and by measuring the cell number in activation culture liquid (especially
Suspension lymphocyte number) and calculate cell density, be compared with certain threshold value set in advance it is such extremely simple
And cheap method, with regard to the propagation of memory T cell and the situation of occupation rate can be grasped.It is most suitable for harvest carefully thus, it is possible to find
The period of born of the same parents carrys out harvesting, and then by the way that the cell freezing of the harvest is preserved, is able to maintain that in memory T cell occupation rate
Simultaneously it is suitably used for the lymphocyte populations of the state added.Further, by by chilled memory T cell group
In carrying out activation culture after defrosting again, high while occupying ratio of memory T cell group is being maintained, cell mass is expanded
Increase, as a result result in the substantial amounts of lymphocyte populations for main component with memory T cell group.In addition, to lymphocyte number and
Incubation time and the amount using memory T cell as the lymphocyte populations of main component finally obtained are studied in detail,
As a result find, manufacture method of the invention, which also has, a large amount of in such short time within 2 weeks after thawing to be manufactured to remember
Recall the effect for the lymphocyte populations that T cell is main component.
In the present invention, to comprising central type memory T cell and effect type memory T cell memory T cell group, by activating
Occupation rate increase is detected caused by culture, the process by carrying out freezing processing, the high pouring of manufacture memory T cell occupation rate
Bar cell mass.Central type memory T cell, which has, goes back to the nest to the function of lymph node, the foreign matter and antigen met head-on in intrusive body, effect
Type memory T cell have in should where position, catch the effect of foreign matter and antigen.Therefore, it is described with memory T cell group
As the adoptive immunotherapy for applying back same organism or it can be bestowed for the lymphocyte populations of main component to the third party
Medical composition use, can expect its be used for than ever adoptive immunotherapy etc., memory T cell occupation rate it is low
Lymphocyte populations have much higher therapeutic effect.
Brief description of the drawings
[Fig. 1] is the 1st during representing activation culture, the lymphocyte of 5,6,13 days is according to whetheing there is cell surface antigen
The figure (n=3) for the result that (CD45RO, CD62L, CD3) is classified.It is used as CD45RO+ present in PBLC
CD62L+ T lymphocytes (CM) and CD45RO+CD62L- T lymphocytes (EM) it is total for memory T cell
(memory) occupation rate at the 5th day, the 6th day be more than 90%, but with culture continue decrease.
[Fig. 2] is to represent that the memory T that occupation rate rises in the cell mass before freezen protective after progress activation culture is thin
Born of the same parents group still maintains the figure (n=3) of high presence ratio after freeze-thaw, during activation culture and amplification cultivation.
[Fig. 3] is the summary using memory cell as the manufacturing process of the medical composition of main component for representing the present invention
Figure.
[Fig. 4] is to represent to the activated lymphocyte group (A) using the existing method making without freezen protective and lead to
The presence ratio for crossing each cell in the activated lymphocyte group (B) of method of the present invention making is based on whetheing there is cell surface antigen
(CD45RO, CD62L, CD3) classified, investigated acquisition result figure." PBMC " represent culture start when, by peripheral blood
The monocyte (component of lymphocyte enrichment) of blood sampling tube separation；"+50 " represent that the 4th day horse back will be added after culture starts
Cell before 50mL culture mediums；"+150 " and " freezing " represent that the 5th day horse back wants the 2nd addition culture medium after culture starts
Or the cell before progress freezing processing；" raw BP " represents further to expand training without freezing process, at once with "+150 "
Cell before the scale of supporting；" raw H " represents the cell for completing above-mentioned raw BP culture, facing before harvest；" TW (defrosting) " is represented
Cell after just frozen cell is thawed；After the completion of " OKT " expression expansion culture, to carry out utilizing OKT3 antibody at once
Carry out the cell before activation culture 2；" defrosting BP " is thin (after activation culture 2) before representing to enter amplification cultivation at once
Born of the same parents；" defrosting H (harvest) " represents the cell after amplification cultivation 2.
Embodiment
" memory T cell " so-called in the present invention, refers to T cell of the expression as the CD45RO of lymphocyte surface antigen,
As the memory T cell, central type memory T cell, effect type memory T cell can be enumerated.Lymphocyte is in its surface table
Up to there is various kinds of cell surface antigen, it can be classified according to surface antigen and be distinguished as a variety of subgroups.For example, lymphocyte can divide
For T cell and B cell, and T cell is differentiated because having CD3 surface antigens on its surface with B cell.Further, as
Surface antigen, the CD45RA antigens for having CD45RO and the xenotype (isoform) as CD45RO are present anti-according to these are whether there is
The classification that original is carried out.CD45RO is (either specific to go back by antigenic stimulus in the initial lymphocyte for expressing CD45RA
Nonspecific) when substitution CD45RA and express, the antigenic type of one of index as memory cell.CD62L is to belong to
The 74kDa of selectin family (selectin family) glycoprotein, expresses in most lymphocyte, is and lymph
Relevant cell adhesion molecule of going back to the nest from cell to lymph node.It also serves as cell surface antigen and is expressed in initial cell, but
With CD45RO expression when, according to CD62L expression intensity, can distinguish central type memory, effect type memory etc. memory cell
Subgroup.Moreover, the T cell of the surface antigen with CD45RO+ and CD62L+ is considered as central type memory T cell, have
The T cell of CD45RO+ and CD62L- surface antigen is considered as effect type memory T cell.
That is, in positive (CD3+) T cells of CD3 that lymphocytic cell surface expresses antigens c D3, cell surface antigen CD45RO
T cell group (CD45RO+, CD62L+) positive positive and CD62L is central type memory T cell group, cell surface antigen
The T cell group (CD45RO+, CD62L-) that CD45RO is positive and CD62L is negative is effect type memory T cell group, these central types
Memory T cell group and effect type memory T cell group collectively form memory T cell group.Inspection to the cell surface antigen can
To be carried out by following methods：Using the antibody through fluorochrome label, (anti-cd 3 antibodies, anti-CD45RO antibody, anti-CD 6 2L resist
Body) lymphocyte is dyed, the fluidic cell such as using FACS Calibur (BectonDickinson company systems)
Instrument (Flow Cytometer) whether there is the fluorochrome labeled antibodies and cell number to measure.In addition it is also possible to through fluorescence
The antibody of dye marker or with first antibody and the secondary antibody through fluorescence labeling is dyed to lymphocyte, swept with laser
Confocal fluorescent microscope is retouched to be observed.
Generally, the occupation rate of memory T cell is 40% or so in the PBLC group of Healthy People, but passes through this hair
Bright manufacture method can improve the occupation rate.As in the present invention " using memory T cell as the lymphocyte of main component
Group " (hereinafter also referred to as " the high lymphocyte populations of memory T cell content "), represents the high lymphocyte populations of memory T cell content
The occupation rate of middle memory T cell be preferably more than 60%, more preferably more than 70%, more than 75%, more than 80%, more than 85%,
More preferably more than 90%, further preferred more than 95% lymphocyte populations.Wherein, memory T cell in lymphocyte populations
Occupation rate is preferably more than 75%.Further, as the lymphocyte populations of the present invention, it is preferable that the 70% of lymphocyte populations
Above, more preferably more than 75%, more than 80%, more than 85%, more preferably 90%, further preferred more than 95% is that central type is remembered
Recall T cell.It should be noted that to be not precluded from memory T thin for the lymphocyte populations using memory T cell as main component of the present invention
T cell beyond born of the same parents is mixed into.
In addition, the present invention using memory T cell as the manufacture method of the lymphocyte populations of main component in, by wrapping successively
Process (a)~(c) high proliferation cultivation is included, the adoptive immunity for being enough to be used in implementing to apply back same organism can be provided
Therapy is bestowed to amount, the lymphocyte populations that memory T cell content is high of the medical composition of the third party for preparing.This
Place, so-called high proliferation cultivation, be can make the lymphocyte number in blood sample breed to more than 500 times, preferably more than 600 times,
More than 700 times, more than 800 times, more preferably more than 900 times of cultural method, by the cultivation, can obtain for example following
Contain the lymphocyte populations of memory T cell with high content, wherein, by 50mL blood obtain using memory T cell as main component
Lymphocyte populations in total lymphocyte number be preferably 5 × 108The individual above, more preferably 8 × 108Individual, further preferred 1 × 109
The individual above, most preferably 3 × 109More than individual.Furthermore, it is possible to obtain wherein using memory T cell as the lymphocyte populations of main component
Cell density be 5 × 104Individual/more than mL, preferably 1 × 105Individual/more than mL, 5 × 105Individual/more than mL, more preferably 1 × 106
The high lymphocyte populations of individual/more than mL memory T cell content.
In addition, as " lymphocyte of collection " in process (a), can enumerate and be come from outside by what conventional method was gathered
All blood or Cord blood and the lymphocyte from marrow, but opportunity from collection, in terms of the easy degree of collection, it is excellent
Select the lymphocyte from peripheral blood.As the peripheral blood, the peripheral blood from other people, the periphery from itself can be enumerated
Blood, during for adoptive immunotherapy, it can be advantageous to use the peripheral blood from itself.From to tumour and its recurrence and
The viewpoint of viral infectious disease, the prevention of autoimmune disease and treatment considers, is moved for receiving organ transplant or marrow
It is preferably the peripheral blood of the supplier from transplant organ or marrow etc. for the patient of plant etc..In addition, being used as the lymph of collection
Cell, preferably uses separated lymphocyte, and the separated lymphocyte can be by using general approach to utilizing
Handled from blood vessel blood sampling, using the peripheral blood (peripheral blood of the preferred acquisition from vein) for singly adopting the collections such as method (pheresis)
To obtain., can be by using sucrose, commercially available lymphocyte separation agent etc. no on separating lymphocyte from peripheral blood
Separation of lymphocytes method known to gradient of continuous density centrifugal process etc. is realized.On the peripheral blood for separating lymphocyte,
It can use and with the addition of heparin or citric acid to prevent the peripheral blood of blood clotting.On the amount of the peripheral blood, do not have
It is particularly limited to, can be suitably set according to manpower and materials, lock out operation of lymphocyte needed for the burden of donor, blood sampling etc.
It is fixed, the amount that single is taken a blood sample can be set as to 0.01mL~100mL or so, more preferably 5mL~50mL or so, further preferably
10mL~20mL.
In addition, the source of lymphocyte is not only defined in people especially, can also enumerate monkey, horse, sheep, goat, pig,
Dog, cat, rat, mouse, hamster, are preferably from people's when the lymphocyte populations of the present invention are bestowed into people.In addition, this hair
Bright lymphocyte populations can be that, from object is bestowed, can be from bestowing the individual beyond object, i.e. donor and acceptor can
With it is identical can also be different.
As " activation culture " in process (a) and process (c), as long as memory T cell can be made by vitro culture
The activation culture that the occupation rate of group rises be (such as known lymphocyte described in Japanese Unexamined Patent Publication 3-80076 publications
Activation culture), it is not particularly limited, is preferably cultivated, more preferably existed in the presence of interleukin 2 (IL-2)
Cultivated in the presence of IL-2 and anti-cd 3 antibodies.For example, can preferably enumerate makes lymphocyte be suspended in containing IL-2's
Culture is with the culture carried out in the culture vessel of anti-cd 3 antibodies in culture medium, in immobilization.It is possible to further according to need
Various division accelerator (mitogen) are used to carry out the activation of lymphocyte.As the anti-cd 3 antibodies, as long as energy
Enough recognize the CD3 surface antigens of lymphocytic cell surface and promote Proliferative Activated antibody, arbitrary AntiCD3 McAb can be used
Antibody.The anti-cd 3 antibodies of activation for stimulating lymphocyte can use purified CD3 molecules in animal or intracellular
Produce, but the excellent commercially available product OKT-3 antibody (manufacturer of stability, handling easiness is advantageously used：EBIO companies).This
Outside, as the activation culture means of the lymphocyte in using memory T cell as the manufacture system of the lymphocyte populations of main component,
The means for being generally used for cell culture can be enumerated, such as cell culture container, cell culture apparatus, cell cultivation groove.
As the cultivation in above-mentioned activation culture, as long as can be with high proliferation after high proliferation cultivation is implemented to complete
Rate manufactures the lymphocyte populations using memory T cell as main component in large quantities, and its condition is not particularly limited.On activation
Culture, because it with the stimulus information of anti-cd 3 antibodies is communicated to lymphocyte and memory T cell group's propagation so as to relative
Premised on the occupation rate for lymphocyte populations rises and then the occupation rate risen is not reduced significantly, so making
For activation culture cultivation period be the lymphocyte for obtaining enough numbers untill, can enumerate 1~10 day, more preferably 2~8
My god, further preferred 3~7 days, particularly preferred 4~6 days.On the replacing frequency of the culture medium in this cultivation period, without spy
Do not limit, in order to prevent nutrient solution from deteriorating and IL-2 activity reductions, carry out within preferably 1~7 day 1 time, preferably with before relative to addition
Nutrient solution in liquid measure for 0.1~5 times or so amount addition.
The activation culture of lymphocyte is carried out in process (a), it is generally, existing in activation culture lymphocyte to be attached to burning
Bottom of bottle face and the cell bred, there is the cell bred after overactivation with the state being suspended in nutrient solution again.Attachment increases
The cell grown and suspension cell are all living cells.Can be culture when nutrient solution starts with activation culture on cultural method
The cultural method of liquid equivalent, can also be that nutrient solution is further added in incubation, continues the activation culture a few days (such as 1
~2 days), further propagation lymphocyte cultural method.The addition of the nutrient solution is preferably when activation culture starts
0.5~2 times of amount of Culture liquid measure, wherein it is possible to preferably enumerate the culture of Culture liquid measure equivalent when starting with activation culture
Liquid.When further adding above-mentioned nutrient solution and proceeding culture, only the lymphocyte that is suspended in nutrient solution can be carried out
Squamous subculture, but it is also possible to which subculture is carried out to the both sides that suspension lymphocyte adds the lymphocyte for adhering to culture vessel
Culture.
In addition, in the activation culture of process (a), lymphocyte number when starting on culture is not particularly limited, but such as
The cell number cultivated in fruit cell culture when starting is very few, then the period extension needed for being raised to cell growth curve, on the contrary,
If excessive, plateau is reached in advance, it is impossible to the method for total amount etc. is increased using addition nutrient solution, so foot can not be obtained
The cell enough measured.It is therefore preferable that inoculation number during to starting is adjusted, so that the growth curve of lymphocyte is raised rapidly,
Above-mentioned T cell group propagation, the ratio occupied in activated lymphocyte promptly rise simultaneously, so as to obtain sufficient amount
Cell.For example, it is preferable to be adjusted to 1.0 × 105~1.0 × 106Individual cell/mL, preferably 1.5 × 105~6.0 × 105Individual cell/
ML, more preferably 4.0 × 105~6.0 × 105Individual cell/mL starts culture again.In addition, capacity and nutrient solution on culture vessel
Amount, it may be considered that operability is suitably selected, and for example, in 225cm2Flask in using 50mL nutrient solutions,
25cm2Flask in use 5mL nutrient solutions example.
As described above, in activation culture in the present invention, on anti-cd 3 antibodies, propagation efficiency, operation from lymphocyte
The viewpoint of easiness consider, preferably used after immobilization.As the support of immobilization anti-cd 3 antibodies, it can make
With the various flasks being made up of materials such as glass, polyurethane, polyolefin, polystyrene, culture dish, culture plate (plate), bag.
Because being readily available, the utensils such as commercially available plastic sterilized cell culture flask can also be used, its size can be with
Suitably selected.On the solidifying methods of above-mentioned antibody, it can also implement and utilize non-specific adsorption or chemical bonding
Method, as long as the solidifying methods that the anti-cd 3 antibodies by immobilization can be stimulated lymphocyte, then can be
Any means.Immobilization can be by the way that the dilution injection of the anti-cd 3 antibodies to be used for carrying out in immobilised utensil, in example
Carried out as stood 2~24 hours at 4~37 DEG C.On the dilution of the anti-cd 3 antibodies, preferably by anti-cd 3 antibodies in warp
In the physiological buffering liquid such as the Du Shi phosphate buffers (Dulbecco ' s Phosphate Buffered Saline) of sterilizing
It is diluted to 1~30 μ g/mL concentration and is used as dilution.Through immobilised utensil until refrigeration can be stored in before use
In room (cold room), refrigerator (4 DEG C), the dilution, the Du Shi phosphate buffers with normal temperature are preferably removed when in use
Cleaned Deng physiological buffering liquid.In addition, as described above, in activation culture in the present invention, from the propagation of lymphocyte
The viewpoint of efficiency considers, interleukin 2 (IL-2) is used preferably in culture fluid nutrient medium.It is preferred that IL-2 is diluted
To make culture be used with the concentration of fluid nutrient medium for 1~2000U/mL.As the IL-2, commercially available product can be used.IL-
2 to be soluble in water, physiological saline, Du Shi phosphate buffers, RPMI-1640, DMEM, IMDA, AIM-V etc. generally extensive
Used in cell culture fluid nutrient medium used etc..In addition, it is once dissolve, then it is preferably stored refrigerated to prevent activity
Reduction.
In addition, as the nutrient solution in above-mentioned activation culture, as long as the nutrient solution of the culture suitable for lymphocyte,
It is not particularly limited, the nutrient solution of the composition containing biological sources such as serum can be used, or added in balance saline solution
The synthetic media of amino acid, vitamin, nucleic acid base etc..Specifically, can enumerate RPMI-1640, DMEM, IMDA,
AIM-V etc., wherein particularly preferred AIM-V.On culture culture medium, the culture medium cultivation effect that with the addition of normal human serum is excellent
It is different, therefore preferably.These culture mediums and human serum can use commercially available product.Culture can come according to common cell culture processes
Carry out.For example, can be in CO2Carried out in incubator (incubator).CO2Concentration is 1~10%, particularly preferably 5%, temperature
For 30~40 DEG C, particularly preferably 37 DEG C, humidity is 90~100%, particularly preferably 95%.
In high proliferation cultivation in the present invention, before and after above-mentioned activation culture, it can also carry out being used to make lymph thin
The culture of born of the same parents' propagation.As the culture, it can enumerate what is carried out under conditions of it there is IL-2 and anti-cd 3 antibodies are not present
Culture (expands culture, amplification cultivation), and its medium component can be adjusted suitably.For example, can enumerate in process (c)
The lymphocyte that has thawed is carried out in the amplification cultivation implemented after activation culture, it is thin to suspension lymphocyte and adhesion lymph
The culture of 4~13 days is passed on, implemented to born of the same parents.In addition, as the nutrient solution in above-mentioned expansion culture, amplification cultivation, as long as
Suitable for the nutrient solution of the culture of lymphocyte, it is not particularly limited, the composition containing biological sources such as serum can be used
Nutrient solution, or add the synthetic media of amino acid, vitamin, nucleic acid base etc. in balance saline solution, can obtain
Obtain commercially available product.Specifically, RPMI-1640, DMEM, IMDA, AIM-V etc. can be enumerated, wherein particularly preferred AIM-V.
In addition, so-called " being detected based on lymphocyte density ... " in process (b), it is right in process (a) to refer to
In the Activated in Vitro culture that lymphocyte is carried out, as needed, until before cell density reaches more than certain threshold value, by
The judgement for proceeding activation culture is made while cell number in secondary measurement nutrient solution.If cell density reaches certain
Threshold value, such as 4 × 105Individual cell/more than mL, then rise to detect the occupation rate of the memory T cell in activation culture liquid.Institute
State the detection based on lymphocyte density, except including the use of microscope to the direct measure of lymphocyte number, use cell count
Beyond the direct measure of device, also including detecting lymphocyte number indirectly under certain cell culture condition.That is, also wrap
Include the detection being carried out as follows：According to the lymphocyte of regulation cell number is cultivated under the conditions of certain activation culture in advance
When cell number determination data, lymphocyte number is speculated indirectly.In addition, when carrying out activation culture to lymphocyte, one
The bottom surface for dividing cell to be attached to culture vessel carries out activation and proliferation, and a part of cell, which is suspended in nutrient solution, is bred.It is another
Aspect, in activation culture, cell number gradually in measurement nutrient solution is while proceed culture on one side.Therefore repeatedly count sometimes
Survey lymphocyte number.If peeling off in this case, will attach to lymphocyte pipette of bottom surface etc., lymphocyte will be peeled off
Uniform suspension is made to be measured with suspension lymphocyte, then the lymphocyte peeled off sometimes increases in stopping after stripping
Grow.Consider that this has problem from the viewpoint of more lymphocytes is harvested, therefore, the measurement to the cell number in culture is preferred
The cell of attachment is not set to be measured to suspension lymphocyte number with peeling off.Due to can consider suspension lymphocyte number and attachment
Lymphocyte number is related to a certain extent to total lymphocyte number, therefore can be calculated by measuring suspension lymphocyte number
The trend of propagation.Therefore, from simplicity easy to operate and the sight for preventing from being generated by it the viewpoint of pollution and breeding from promotion
Put to consider, measurement suspension lymphocyte is useful.In addition, considering from viewpoints such as yields, lead to preferably at the end of culture
Cross and pat the cell that will attach to culture vessel bottom surface and also strip down, total lymphocyte number is measured.
Therefore, so-called " occupation rate of memory T cell group rises " in process (b), refers to train through overactivation in process (a)
The rising for occupying ratio of memory T cell in the sum of foster lymphocyte, specifically, the occupation rate of memory T cell reaches
To such as more than 80%, preferably more than 85%, more than 90%, more preferably more than 95%.The occupation rate of the memory T cell can also
Directly the cell surface antigens such as CD3, CD45RO, CD62L are investigated using antibody by using flow cytometer to calculate.
In addition, as it is in using memory T cell as the manufacture system of the lymphocyte populations of main component, based on lymphocyte Density Detection
The means that the occupation rate of memory T cell group rises, can enumerate the means for being generally used for determining lymphocyte number, for example, suspend and drench
Cell counter of bar cell etc..
" freezen protective " in process (b) can be carried out according to common cell freezing method.For example, can be by lymph
Cell suspension is protected in cell freezings such as Bambanker (Lymphotec company systems), CP-1 (Jidong Pharmaceutical Industry Co., Ltd.'s system)
In liquid storage product and it is stored in freeze storage container, the container is put into and freezes cup (BICELL) (Japanese Freezer companies
System) in, temporary safe-keeping moves into liquid nitrogen container (MVE) in -80 DEG C of ultra low temperature freezer (Sanyo Electric Co., Ltd's system) after 2 days
In, it is stored in as Cell Cryopreservation in liquid nitrogen.In addition, being raised from the growth curve in the activation culture after freeze-thaw
Viewpoint consider that the lymphocyte number preserved in 1 freeze storage container of preferred pair is adjusted, preferably every 1 freezing is protected
Deposit and 1 × 10 is preserved in container7~5.0 × 107Individual cell.The cell being stored in multiple freeze storage containers can be merged,
For the culture after freeze-thaw, but consider from the easiness of operation and the viewpoint of qualitative control, preferably protect 1 freezing
Deposit the lymphocyte in container and move to and cultivated in the culture vessel of 1 or more than 2.In addition, as using memory T cell as
The freezen protective means of lymphocyte in the manufacture system of the lymphocyte populations of main component, can enumerate be normally used for it is cold
Freeze preserve cell means, such as freeze storage container, freeze cup, ultra low temperature freezer, liquid nitrogen container.
In process (c) to " defrosting " of lymphocyte that has been frozen can according to common frozen cell defreezing method
Come carry out.For example, can be added by using 37 DEG C dry type heating unit (Dry thermo unit, Taitec company system) etc.
4 minutes are heat-treated to be thawed.It is preferred that by the lymphocyte that has thawed is moved into appropriate culture medium after
Centrifugally operated is carried out at room temperature, remove supernatant to remove cell freezing preservation liquid.In addition, thin on the lymph after the defrosting
The activation culture of born of the same parents, can also continue in the absence of AntiCD3 McAb resist after the activation culture in the presence of anti-cd 3 antibodies
Amplification cultivation under the activation stimulation of body.I.e., it is possible to continue the utensil in non-immobilization anti-cd 3 antibodies (for example, culture is burnt
Bottle, rolling bottle (roller bottle), gas permeability culture bag etc.) in proceed culture until obtaining substantial amounts of cell.Now,
Carried out preferably in the presence of interleukin 2 (IL-2).On the culture of the lymphocyte under above-mentioned condition, except no AntiCD3 McAb
Beyond the stimulation of antibody, concentration of human serum etc. is identical preferably with there is the condition for the culture that activation is stimulated, from operability, cost
Property, in terms of security from the point of view of, more preferably in time use serum free medium.In addition, as using memory T cell as it is main into
Point lymphocyte populations manufacture system in the defrosting means for the lymphocyte being frozen, can enumerate be generally used for freezing
The means of the defrosting of cell, such as dry type heating unit.
According to the present invention using memory T cell as the manufacture method of the lymphocyte populations of main component, result in and include
, more than 85%, more preferably more than 90%, more than 75%, preferably more than 80% further preferred more than 95% memory T cell
Lymphocyte populations, comprising more than 70%, more preferably more than 75%, more than 80%, more than 85%, more preferably 90%, it is further excellent
Select the lymphocyte populations of more than 95% central type memory T cell.In addition, the present invention can be made containing above-mentioned thin with memory T
Born of the same parents for main component lymphocyte populations as active ingredient, for adoptive immunotherapy or for bestowing to the third party's
Medical composition.The medical composition may serve as antitumor agent, HIV therapy medicine, viral infectious disease medicine, from
Surviving for transplanted cells or organ after body immunity disease medicine, cell or organ transplant promotes medicine, prevention of tumor
The medicine of recurrence.In addition, ratio of the lymphocyte populations using memory T cell as main component of the present invention due to memory T cell group
Rate is up to more than 80%, it is possible to do not implement the antibody of surface antigen for using the cell surface for lymphocyte populations to exist
Etc. numerous and diverse separation concentration operations such as memory T cell group are separated, and suspension obtained by suitable solution is suspended in
It is used as medical material.As the present invention medical composition form, preferably it is non-orally, be preferably suspended this hair
The liquids such as injection, the point drops of bright lymphocyte populations.Especially, more preferably the lymphocyte populations of the present invention are suspended in
Injection, point drops in transfusion physiological saline that with the addition of human serum albumins with 0.01~5% amount etc., but do not limit
Due to this.It is preferably the drop to vein or the injection to vein, artery, part etc. as method is bestowed.The liquid measure root bestowed
According to the different and different of the position bestowed method, bestowed, it is often preferred that 1~500mL, preferably make in this liquid measure comprising hereafter institute
The lymphocyte number for the lymphocyte dosage stated.In addition, in the medical composition of the present invention, except containing memory T cell content
Beyond high lymphocyte populations, it can also contain stabilizer, buffer components, other treatment medicine, replenishers etc..On the present invention
Medical composition the amount of bestowing, bestow number of times, bestow period etc. and the present invention medical composition in lymphocyte populations
Concentration, be not particularly limited, can suitably be adjusted according to sign, the state of an illness, body weight, age, sex for bestowing object etc.,
In normal circumstances, preferably with 1 × 102Individual~1 × 109Set on the basis of individual relative to the every 1Kg body weight for bestowing object
The lymphocyte amount of bestowing.For further effectively, preferably 1 × 10 will be set as per the lymphocyte amount of bestowing of 1Kg body weight relatively3It is individual
More than, even if but being due to be more than 5 × 108The individual effect that can not predict further increases, so most preferably will be per 1Kg body weight
The amount of bestowing be set as 1 × 103Individual~5 × 108It is individual.On bestowing frequency, 1 times/day~1 time/month can be preferably enumerated, this
Outside, number of times is bestowed to be necessary at least more than 1 time.The present invention it is medical composition in use, can also and with others hands
Art, drug treatment.
Embodiment given below, specifically describes the present invention, but the present invention is not limited to following record.
Embodiment 1
[activation and proliferation of lymphocyte]
(1) preparation of PBLC
From each 50mL of peripheral blood of the Healthy People of informed consent of venous collection 3 in the way of adding heparin.To respectively it adopt
Blood test sample moves to 250mL centrifugal precipition tube (BD Falcon；352075) in, 100mL is added in every part of test sample
Cleaning solution (with the addition of the physiological saline of 0.1% human albumin：Physiological saline 500mL, manufacturer：Great mound pharmacy,
The people of 2.0mL 25% donate blood albumin, manufacturer：The pharmacy of Tian Bian Mitsubishis), mix at leisure, dilute 3 times.Including this operation
Following operation aseptically carry out.For every 1 part of test sample, in 4 ficolls for being filled with 15mL (Ficoll)
50mL centrifugal precipition tubes (the BD Falcon of (GE Healthcare Bioscience company systems)；352070) make in dilute through 3 times
The blood double-layer released, by the centrifugal precipition tube at room temperature with 20 minutes (centrifuges of 1800rpm continuous centrifugals：Co., Ltd.
KOKUSAN systems；H-700).The mononuclear cell layer in intermediate layer is recycled to the 50mL centrifugations for being previously implanted cleaning solution
Guan Zhong.The lid of centrifugal precipition tube is covered, mixing 2~3 times is overturned, is centrifuged 15 minutes with 1800rpm at room temperature.Removed after centrifugation
Supernatant, disperses sediment, then adds cleaning solution, has obtained the cell suspension that total amount is 50mL.Will be described mixed
Suspension under normal temperature with 1800rpm in being centrifuged 10 minutes, and the culture medium 1 that 50mL is added in obtained sediment (contains 2% human blood
(Lymphotec company systems) and 350U/mL rIL-2 (Proleukin clearly：Novartis company systems) AIM-V culture mediums
(Invitrogen company systems)), it is suspended, has obtained cell suspension.
40 μ L your the kirschner solution (Turk ' s solution) (Wu Teng Chemical Companies system) that carries is injected into 5mL circles
Bottom test tube (BD Falcon；352008) in, the 10 above-mentioned cell suspensions of μ L being added thereto and are mixed, 10 μ L mixed liquors are filled
Enter Neubauer types blood cell counting plate (Elmer company systems；9731), in microscope (Olympus optics industrys Co., Ltd.
System；211320) cell number has been calculated under.
In addition, by 20 μ L Trypan Blue liquid (SIGMA company systems；93595) it is injected into 5mL round bottom test tubes (BD
Falcon；352008) in, the 10 above-mentioned cell suspensions of μ L are mixed thereto, and it is thin that 10 μ L mixed liquors are poured into Neubauer type blood
Born of the same parents' tally, measures the living cells not being colored, has calculated survival rate under the microscope.
(2) preparation of OKT3 immobilizations flask
With physiological saline by OKT3 (import retailers：Janssen Kyowa Co., Ltd., manufacturer：Ortho
Pharmaceutical：OKT3 is noted) preparing solution is 5 μ g/mL, and 10mL seasoning liquids are injected into floor space for 225cm2Culture
With flask (Sumitomo Bakelite Co. Ltd. systems；MS-2180R in), bottom surface is made all to be covered by OKT3 solution.Stand 3 hours
More than, the OKT3 solution in flask then is drawn with attractor, about 100mL physiological saline (manufacturer is injected：The pharmacy of great mound),
Flask lid is covered, acutely vibration, then discards liquid therein.Again by about 100mL physiological saline (manufacturer：Great mound systems
Medicine) pour into flask, make immobilization down, stand 15 minutes at room temperature.Thereafter, close the lid, acutely vibration 1 minute, is abandoned
Remove liquid therein.Carefully drawn with attractor remain in flask and bottle cap on liquid, obtained OKT3 immobilizations burning
Bottle.If on the day of preparation without using, retain a small amount of physiological saline, until use before be stored in 4 DEG C.
It is 25cm for floor space2Culture flask (Sumitomo Bakelite Co. Ltd. systems；MS2105R), also with it is upper
State record and similarly implement immobilization processing.
(3) to the research -1 of inoculation lymphocyte number
The cell suspension prepared in (1) is centrifuged 15 minutes with 800rpm at room temperature.With culture medium 1 by cell density
0.2 × 10 is allocated as respectively5Individual/mL, 1.0 × 105Individual/mL, 2.0 × 105Individual/mL, 2.0 × 106Individual/mL, 6.0 × 106Individual/
mL.The cell suspension of each cell density is injected separately into the OKT3 immobilizations for being seeded to and being prepared in above-mentioned (2) with each 5mL
25cm2In bottle, with culture medium 1 in TE-HER types CO2With 37.0 ± 0.5 DEG C of temperature, humidity in incubator (HIRASAWA company systems)
95.0 ± 5.0%, CO2Concentration 5.0 ± 0.2% is cultivated.The method meter same with (1) is suitably used in the training period
Suspension cell number and survival rate are calculated, nutrient solution is observed with the color change of vegetative state while suitably addition training
Support base 1, continuous culture 10 days.Also, at the end of culture, culture flask is patted, the lymphocyte that will attach to bottom surface also enters
Row is reclaimed, and total lymphocyte number and survival rate have been calculated with the method measurement same with (1).
As a result：
As shown in Table 1, inoculating cell density is 0.2 × 105During individual/mL, even if culture can not obtain enough for 10 days
Cell number, in addition, being 6 × 106During individual/mL, hypoproliferation, survival rate are also bad, thus determined both inoculating cells close
Degree is improper.Further, less than 90% inoculating cell density 1.0 × 10 will not be turned into survival rate5Individual/mL and 2.0 ×
106Between individual/mL, inoculation cell density is made further research.In addition, in this research, with the color change of nutrient solution
Culture medium 1 has suitably been added for index with suspension cell number, in order that operation is easier, realize more rapid cell propagation,
Need to determine the period of adding of culture medium from cell density and cultivated days, therefore period added to nutrient solution to be ground
Study carefully.
The change of the inoculating cell density of table 1 and propagation and survival rate
(4) research in period is added to nutrient solution
According to the step same with (1), PBLC is prepared from the Healthy People of 3 informed consents, and measure
Cell number.
The lymphocyte of preparation is centrifuged in the same manner as (3), cell density is allocated as 0.5 respectively with culture medium 1
×105Individual/mL, 1 × 105Individual/mL, 2.0 × 105Individual/mL, 4 × 105Individual/mL, 8 × 105Individual/mL.By the cell of each cell density
Suspension is injected into the OKT3 immobilizations 225cm prepared in above-mentioned (2) with each 50mL2Flask (Sumitomo Bakelite Co., Ltd.
System；MS-2180R in), with culture medium 1 in TE-HER types CO2With temperature 37.0 ± 0.5 in incubator (HIRASAWA company systems)
DEG C, humidity 95.0 ± 5.0%, CO2Concentration 5.0 ± 0.2% starts culture.Consider from the viewpoint of culture efficiency, in culture
Start to have added 50mL nutrient solutions into each flask at the 3rd day, using the method same with (1), counted before nutrient solution is added
Suspension cell number has been surveyed, total cell number has been measured when adding (the 4th day) next day recovery cell.
As a result：
As shown in Table 2, inoculating cell number is 2.0 × 107(4.0×105Individual/mL) or 4.0 × 107(8.0×105Individual/mL)
When, there is the test sample that proliferation rate (obtained by total cell number divided by inoculating cell number after culture) is more than 2.5 times.That is,
The 4th day (1.1 × 10 of test sample 18It is individual and 1.3 × 108It is individual) and the 4th day (1.0 × 10 of test sample 38It is individual).Adding
Plus the total cell number obtained after nutrient solution is more than 108Test sample in, suspension of the test sample before nutrient solution is added is thin
Born of the same parents' number is 1.9 × 107It is individual and 6.3 × 107(the 3rd day of test sample 1) and 2.0 × 107Individual (the 3rd day of test sample 3).
Therefore, set after culture starts the 3rd day, reach 3.8 × 10 in suspension cell number5Individual/more than mL or 4.0 × 105Individual/
Nutrient solution is added during mL.
Table 2
(5) to the research -2 of inoculation lymphocyte number
According to the step same with (1), PBLC is prepared from the Healthy People of 3 informed consents, and measure
Cell number.
Lymphocyte in the same manner as research -1 with the inoculation lymphocyte number of (3) to preparation centrifuged, with culture medium 1
Cell density is allocated as 1.0 × 10 respectively5Individual/mL, 1.5 × 105Individual/mL, 2.0 × 105Individual/mL, 4.0 × 105Individual/mL, 6
×105Individual/mL, 8 × 105Individual/mL, the OKT3 immobilizations prepared in above-mentioned (2) are injected into by each cell suspension with each 5mL
25cm2In flask, with culture medium 1 in TE-HER types CO2With 37.0 ± 0.5 DEG C of temperature, wet in incubator (HIRASAWA company systems)
Degree 95.0 ± 5.0%, CO2Concentration 5.0 ± 0.2% is cultivated.Gradually measured with the method same with (1) after culture starts
Suspension cell number and living cell rate, 4 × 10 are changed into cell density55mL nutrient solutions are added during individual/more than mL in each flask,
Amount to most long cultivated 6 days.At the end of culture, the lymphocyte for being attached to bottom surface is handled in the same manner as (3), gathered
Whole cells, total cell number and survival rate have been calculated with the method measurement same with (1).
As a result：
In this research, activation culture will be carried out in most short number of days and most cells are obtained to be used as target.
Inoculating cell density is 1.0 × 105During individual/mL, even in culture the 5th day, 2 surveys in 3 test samples
Suspension cell number is not up to 4 × 10 in test agent5Individual/mL.It is 1.0 × 10 in inoculating cell density5Individual/mL test sample
3、1.5×105Individual/mL test sample 1~3,2.0 × 105Individual/mL test sample 1~3 and 4.0 × 105Individual/mL's
In test sample 1, suspension cell density has exceeded 4 × 10 within the 5th day5Individual/mL, has added 5mL nutrient solutions, as a result next day the (the 6th
My god) total cell number exceeded 1.2 × 10 mostly7.Inoculating cell density is 4.0 × 105Individual/mL test sample 2 and 3,6.0 ×
105Individual/mL test sample 1~3 and 8.0 × 105In individual/mL test sample 1~3, at the 4th day, suspension cell was close
Degree is more than 4 × 105Individual/mL, total cell number exceedes in the next day of the 5th day for having added nutrient solution, above-mentioned test sample
1.2×107It is individual.
In summary, inoculating cell density when culture starts is 1.5 × 105During individual/mL, at the 5th day more than 4 × 105
Individual/mL and culture is completed in next day；For 4.0 × 105During individual/mL, it was more than about 4 × 10 at the 4th day5Individual/mL and in next day complete
Culture；For 6.0 × 105Individual/mL and 8.0 × 105During individual/mL, at the 4th day then all more than 4 × 105Individual/mL and in next day
Complete culture.Thus, it is believed that in order to which during shortening culture, inoculating cell density is set as into 4.0 × 105Individual/more than mL,
8.0×105Individual/mL is the following is suitable.
The relation of cell number and propagation when the culture of table 3 starts
n/a：It is unavailable
Embodiment 2
[change of lymphocyte surface antigen during activation culture]
(1) preparation of lymphocyte and activation culture
According to the step same with (1) of embodiment 1, periphery hemolymph is prepared from the Healthy People of 3 informed consents thin
Born of the same parents, and measured cell number.
In the same manner as (3) of embodiment 1, the lymphocyte of preparation is centrifuged, with culture medium 1 by the thin of lymphocyte
Born of the same parents' density is allocated as 6 × 105Individual/mL.The 50mL cell suspensions are injected into the 225cm made in (2) of embodiment 12OKT3
Immobilization flask (Sumitomo Bakelite Co. Ltd. systems：MS-2180R in), existed in the same manner as (3) of embodiment 1 with culture medium 1
TE-HER types CO2With 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ± 5.0%, CO in incubator (HIRASAWA company systems)2Concentration
5.0 ± 0.2% are cultivated.During culture, suspension cell is gathered at any time and suspension cell is measured in the same manner as (1) of embodiment 1
Number.Due to turning into 4 × 10 within the 4th day in culture5Individual/more than mL, so adding 50mL culture medium 1, and proceeds culture.
Cultivate the 5th day and add 50mL culture medium 1 again, and proceed culture.In culture the 6th day, make to be attached to admittedly by patting
The lymphocyte of the bottom surface of phaseization flask is peeled off, and has obtained cell suspending liquid.By 25mL, the cell suspending liquid is injected into non-solid phase
Change OKT3 225cm2In bottle, further addition 75mL culture medium 1, and proceed culture.Added within the 9th day in culture
100mL culture medium 1, and culture is continued to the 13rd day.Make to be attached to flask bottom surface by patting within the 13rd day in culture
Lymphocyte peel off, reclaimed as cell suspending liquid, complete culture.
During this period, in cultivate start when, culture starts the 5th day (to add at once before culture medium), (attachment is thin within the 6th day
Born of the same parents peel off front/rear) and the 13rd day, a part of cell suspending liquid is gathered, the surface antigen of the lymphocyte of collection is entered immediately
Row analysis.It should be noted that known now lymphocyte almost 100% be CD3 positive cells (non-Patent Citation 3).
(2) analysis method of lymphocyte surface antigen
The cell suspending liquid of flow collection according to above-mentioned (1) is respectively taken into 5mL, 15mL centrifugal precipition tubes (BD is placed in
Falcon；352097) in, centrifuged 5 minutes with 1800rpm at room temperature.Albumin sheath fluid is added into obtained sediment
(sheath fluid) (Beckman Coulter company systems：IsoFlow), each cell suspension is allocated as 1.0 × 107Individual/
mL.Be injected separately into 5mL round bottoms test tube (BD Falcon) 10 μ L through fluorochrome label each antibody (anti-cd 3 antibodies,
BectonDickinson company systems 349201；Anti- CD45RO antibody, with 340438；Anti-CD 6 2L antibody, Becton
DickinsonPharmingen company systems 555544), above-mentioned each each 50 μ L of cell suspension are then injected into each test tube
In, reaction 30 minutes is allowed to rest under conditions of shading, 4 DEG C.2mL albumin sheath fluids are added in each test tube after the reaction,
Centrifuged 5 minutes with 1800rpm at room temperature.Attract to remove supernatant, 500 μ L CellFix is added into each sediment
Suspension is made in (BectonDickinson company systems), for target antibody Staining Lymphocyte group, uses flow cytometer
(BectonDickinson company systems；FACS Calibur) it is determined, the cell number of measure is 10,000.
(3) analysis rolled into a ball to antibody Staining Lymphocyte
The antibody staining lymphocyte group of making is analyzed according to the method for above-mentioned (2).
As a result：
It typically, there are the T lymphocytes of the CD45RO+CD62L+ in PBLC and CD45RO+CD62L- T
The ratio of lymphocyte is 20~40% or so (tables 4).But, by implementing activation culture, at the 5th day, the occupation rate exceeded
90%th, at the 6th day more than 95%.But further acknowledged that if continuation is cultivated, the occupation rate risen would be reduced to initial level
(about 40%) (table 4, Fig. 1)." initial (Naive) " represents CD45RO-, CD62L+, CD3+ T cells, " effect
(Effector) CD45RO-, CD62L-, CD3+ effector T cell " are represented, " CM " is represented in CD45RO+, CD62L+, CD3+
Centre type memory T cell, " EM " represents CD45RO+, CD62L-, CD3+ effect type memory T cell, is used as " CM+EM " or " memory
(memory) " represent CD45RO+, CD62L+/-, CD3+ memory T cell.
Therefore, it is possible to confirm that the suspension cell density in activation culture turns into 4 × 105Individual/more than mL, culture is added
After the next day of base, the occupation rate of CD45RO+, CD62L+T lymphocyte and CD45RO+, CD62L-T lymphocyte rises.Cause
This, in order to increase the memory cell number of harvest, it is determined that turn into 4 × 10 in suspension cell density5Individual/more than mL, training is added
After the next day of foster base and stop activation culture before memory T cell occupation rate is reduced, make to be attached to burning by patting
The lymphocyte in bottom of bottle face is peeled off, makes into suspension cell, reclaims whole cells.
The change (%) of the occupation rate of lymphocyte surface antigen during 4 activation culture of table
Embodiment 3
[research to the frozen cell density in freezing process]
Preparation containing the T cell group obtained by the method for the present invention will be used to treat, the lymphocyte finally obtained
Quantity more than it is just efficient.Activation amplification cultivation when therefore, on freezen protective after the cell number influence of relatively every 1 test tube
Possibility is studied.
(1) to the activation culture of research -1 of frozen cell density
According to the step same with (1) of embodiment 1, periphery hemolymph is prepared from the Healthy People of 3 informed consents thin
Born of the same parents, and measured cell number.
In the same manner as (3) of embodiment 1, the lymphocyte of preparation is centrifuged, with culture medium 1 by the thin of lymphocyte
Born of the same parents' density is allocated as 6 × 105Individual/mL.The 50mL cell suspensions are injected into the 225cm made in (2) of embodiment 12OKT3
Immobilization flask (Sumitomo Bakelite Co. Ltd. systems：MS-2180R in), in TE-HER types in the same manner as (3) of embodiment 1
CO2With 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ± 5.0%, CO in incubator (HIRASAWA company systems)2Concentration 5.0 ± 0.2%
Cultivated.Exceed 4 × 10 due to starting the 3rd day suspension cell number in culture5Individual/mL, so addition 50mL culture medium 1
And continue culture to next day.Stop culture within the 4th day what culture started, by patting the lymphocyte for making to be attached to flask bottom surface
Peel off, become suspension cell, reclaim whole cells, obtained coming from the cell suspension of culture medium 1.According to embodiment
1 (1) same step has measured the cell number in above-mentioned suspension.
(2) to the freezen protective of research -2 of frozen cell density
Based on the cell density of measurement in above-mentioned (1), for 3 kinds of test samples, culture medium 1 is added so that relatively every 1
The lymphocyte number of cryopreservation tube (Corning company systems) is allocated as 0.5 × 107It is individual, 1.0 × 107It is individual, 3.0 × 107It is individual, 5.0
×107It is individual, it is injected into 15mL centrifugal precipition tubes, is centrifuged 5 minutes with 1200rpm at room temperature.After centrifugation, each centrifugation is removed heavy
Supernatant in the pipe of shallow lake, so as to get sediment disperse, addition cell freezing preserves liquid (CP-1：Extremely eastern pharmaceuticals industry strain formula meeting
Society's system), the liquid measure for making every 1 cryopreservation tube is 1.8mL, by cell suspension and is injected into cryopreservation tube, the cryopreservation tube is inserted into jelly
Deposit in cup (Japanese Freezer company systems), the temporary safe-keeping 2 in -80 DEG C of ultra low temperature freezers (Sanyo Electric Co., Ltd's system)
My god, then move into liquid nitrogen container (MVE), preserved as Cell Cryopreservation.
(3) defrosting of research -3 to frozen cell density is handled
It will be taken out, heated in the dry type for being set as 37 DEG C single from liquid nitrogen container with the cryopreservation tube of each cell density freezen protective
Heating 4 minutes in first (Taitec company systems), thaw to freezing lymphocyte.In 1 pair of defrosting of culture medium with 10mL
Lymphocyte move it into 15mL centrifugal precipition tubes while be suspended, centrifuged 3 minutes with 1200rpm at room temperature.
After centrifugation, supernatant is removed, makes scattered sediment, addition 10mL culture medium 1, cell suspension be made, with embodiment 1
(1) cell number has similarly been measured.On the lymphocyte thawed, because cell number is different in every pipe, if so directly
Suspension inoculation is carried out with the culture medium of same amount, then there is the differentia influence propagation of inoculating cell number.In order to exclude this
Possibility, in the inoculation cell thawed and the process cultivated, in before inoculation and each in process to lymphocyte number
Measured, cell density during on inoculation makes cell density substantially certain by adjusting the Culture liquid measure of addition certainly
(7.5×105Individual/mL).Also used in follow-up expansion culture, activation culture, amplification cultivation cultivate start when to because
Cryopreservation tube is different and method that different cell number carries out liquid measure regulation.The Culture liquid measure in each operation is shown in table 5 and is added
Liquid volume added.
As operation when thawing, the above-mentioned suspension of the liquid measure shown in table 5 is seeded to flat 24 hole with each 2mL in every hole
On porous plate (plain 24-well multi-well plates) (Sumitomo Bakelite company systems), day inoculation is regard as the 1st
My god, 37 DEG C, humidity 95%, CO2Culture 2 days in the CO2gas incubator of concentration 5.0%.
The amount of the culture medium 1 of Culture liquid measure and addition in each operation of table 5
(4) culture and activation culture are expanded
The lymphocyte that will be cultivated in (3) after defrosting is handled is in the 3rd day respectively from 24 hole porous plates
Move in boiling flask, with carried out in the hole of 1 pair of 24 hole porous plate of culture medium of each addition liquid measure described in table 5 cleaning so as to
The cell of residual is reclaimed, the liquid measure in flask is deployed into the liquid measure described in table 5.By each flask in TE-HER types CO2Culture
With 37.0 ± 0.5 DEG C of temperature, humidity 95%, CO in case (HIRASAWA company systems)2Concentration 5.0% further culture 2 days (table 5,
Expand culture).
After above-mentioned expansion culture, in order to carry out activation culture, the cell suspension in culture flask is moved to and real
In the OKT3 immobilization flasks of capacity shown in apply that (2) of example 1 be prepared as, table 5, burnt with 1 pair of culture of culture medium
The inside of bottle is cleaned, and reclaims the cell of residual, and the most liquid measure at last in OKT3 immobilizations flask is allocated as described in table 5
Liquid measure, by each flask in TE-HER types CO2In incubator (HIRASAWA company systems) with 37.0 ± 0.5 DEG C of temperature, humidity 95%,
CO2Concentration 5.0% further cultivates 2 days (table 5, activation culture 2).
After above-mentioned activation culture, patting each OKT3 immobilizations flask peels off the cell for being attached to bottom surface, obtains
Cell suspension.By new flat culture flask (the Sumitomo Bakelite companies described in obtained cell suspension immigration table 5
System) or new gas permeability culture bag (1000mL, Kohjin Bio company systems 16087528) in, further with shown in table 5
The culture medium 2 for adding liquid measure (contains 1% human serum (Lymphotec company systems), 175U/mL rIL-2 (Proleukin：
Novartis company systems) AIM-V culture mediums：Invitrogen systems) to being cleaned in each flask, the cleaning fluid is merged
Enter in the cell suspension in the boiling flask or gas permeability culture bag, in TE-HER types CO2Incubator (HIRASAWA companies
System) it is interior with 37.0 ± 0.5 DEG C of temperature, humidity 95%, CO2Concentration 5.0% further cultivates 3 days (table 5, amplification cultivation 1).
After above-mentioned amplification cultivation 1, the culture medium 2 of the addition liquid measure described in table 5 is added in above-mentioned culture vessel,
37 DEG C, humidity 95%, CO2Culture 4 days is continued in the CO2gas incubator of concentration 5.0%.Herein, newly supplement
When flask is as culture vessel, the culture medium 2 of addition addition liquid measure, vibrates flask and to cell suspension in the flask into culture
Liquid is stirred, and the half amount of capacity is moved into new flask, proceeds culture.As culture vessel, moved to from flask
When in bag, into 2 flasks, the culture medium 2 of injection addition liquid measure, vibrates flask and cell suspension is stirred, by content
The total amount of amount is moved into new bag, proceeds culture.When newly-increased adduction uses culture bag as culture vessel, what is newly used
The culture medium 2 of addition liquid measure in culture bag described in addition table 5, it is sterilely bonded in above-mentioned amplification cultivation 1
In the culture bag cultivated, the content in two bags is sufficiently mixed, continues to be cultivated (table 5, amplification cultivation
2)。
When carrying out each operation described in (3) and (4), measurement has calculated each test specimens in the same manner as (1) of embodiment 1
The cell number and survival rate of product.It the results are shown in table 6.
As a result：The cell number being housed in cryopreservation tube is adjusted to each setting value to carry out freezen protective, throughout science and engineering
Do not observe there is big difference between 3 test samples in sequence.But, the memory T lymph manufactured in the method for the present invention is thin
Born of the same parents group will be used to treat, and consider from the viewpoint of efficiency, and the cell mass more than cell number obtained by a series of culture is to make us
It is desired.Therefore, because appropriate culture scale is expected, it is believed that the cell number for a relative cryopreservation tube is suitably for
1.0×107Individual~5.0 × 107It is individual.In the process of the present embodiment (1)~(4), culture is increased in order to exclude inoculating cell density
The influence grown, as shown in table 5, set-point is adjusted to annex solution by cell density.On the other hand, by the training of amplification cultivation 1
Support cell number when starting and be set as 1 × 108It is individual, 2 × 108It is individual, 3 × 108It is individual, liquid measure when starting culture for set-point simultaneously
Cultivated, it is 1 × 10 to be as a result able to confirm that cell number8Amplification delay about 2 days when individual, and it is 2 × 108It is individual and 3 × 108It is individual
When, then the memory T lymphocytes of sufficient amount are obtained after the process of above-mentioned amplification cultivation 2 without significant difference
Group.If the cell number for an above-mentioned relative cryopreservation tube is set as into 1.0 × 107~5.0 × 107It is individual, then it can meet this
Condition.
Cell number, survival rate and cell density (measured value) after the defrosting treatment process of table 6
Embodiment 4
[change that the lymphocyte surface antigen in the process of activation amplification is carried out after chilled preservation]
(1) making of Cell Cryopreservation
In embodiment 2, it specify that：In activation culture before cell freezing, the cell density in nutrient solution turns into 4
×105In individual/more than mL stage, the possession ratio of the memory T cell group in the cell in nutrient solution rises.
Therefore, in the same manner as (1) of embodiment 3, according to the step same with (1) of embodiment 1, obtained same from 3
The Healthy People of meaning prepares PBLC, and the cell number of the cell suspension to obtaining is measured, adjusts cell density
With for 6 × 105Individual/mL, its 50mL is injected into and burnt according to the OKT3 immobilizations made with (2) of embodiment 1 same step
Bottle 225cm2In, in TE-HER types CO2In incubator (HIRASAWA company systems) with 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ±
5.0%th, CO2Concentration 5.0 ± 0.2% is cultivated.Exceed 4 × 10 due to starting the 3rd day suspension cell number in culture5Individual/
ML, so the culture risen as the possession ratio of memory T cell group, adds 50mL culture medium 1 and continue culture extremely
Next day.Stop activation culture in next day (i.e. culture starts the 4th day), the lymphocyte for being attached to flask bottom surface is shelled by patting
From, become suspension cell, reclaim whole cells, and cell suspension has been made with culture medium 1.According to embodiment 1
(1) same step is measured to the cell number and survival rate in above-mentioned suspension, will be equivalent to 7.0 × 107Individual cell
The suspension of amount is moved into 50mL centrifugal precipition tubes, is centrifuged 5 minutes with 1200rpm at room temperature.Remove in centrifugal precipition tube
Supernatant, the CP-1 cell freezings that 3.6mL is added into sediment preserve liquid (extremely eastern pharmacy system), and are suspended.In 2 jellies
Deposit and injected in pipe each 1.8mL of above-mentioned suspension, freezen protective is carried out in the same manner as (2) of embodiment 3.
(2) defrosting to Cell Cryopreservation is handled
By Cell Cryopreservation in being taken out after 1 week from liquid nitrogen container, research with (3) frozen cell density of embodiment 3-
3 have similarly carried out defrosting processing.Heating 4 minutes, right in the dry type heating unit (Taitec company systems) for being set as 37 DEG C
Freezing lymphocyte is thawed.Moved it into while suspension with 10mL 1 pair of lymphocyte thawed of culture medium
In 15mL centrifugal precipition tubes, centrifuged 3 minutes with 1200rpm at room temperature.After centrifugation, supernatant is removed, sediment disperseed, added
Plus 40mL culture medium 1, cell suspension is made, measure cell number in the same manner as (1) of embodiment 1.It should be noted that
Know the lymphocyte after freezen protective almost 100% be CD3 positive cells (non-Patent Citation 1).
(3) the expansion activation amplification cultivation of Cell Cryopreservation
With each 2mL in every hole, the suspension is seeded to flat 24 hole porous plate (Sumitomo in the same manner as (4) of embodiment 3
Bakelite company systems) on, will inoculation day as the 1st day, 37 DEG C, humidity 95%, CO2The carbon dioxide training of concentration 5.0%
Support culture 2 days in case.By the lymphocyte through culture in the 3rd day from 24 hole porous plates move to boiling flask, with 1 pair of culture medium
Cleaning is carried out in the hole of 24 hole porous plates so as to reclaim the cell of residual, the liquid measure of the cell suspension in flask is deployed into
90mL.By above-mentioned flask in TE-HER types CO2With 37.0 ± 0.5 DEG C of temperature, humidity in incubator (HIRASAWA company systems)
95%th, CO2The further culture 2 days (expanding culture) of concentration 5.0%.After culture is expanded, in order to carry out activation culture, it will train
The cell suspension supported in flask moves to 225cm2OKT3 immobilization flasks in, with 1 pair of culture flask of culture medium
Portion carries out cleaning so as to reclaim the cell of residual, and the most liquid measure at last in OKT3 immobilizations flask is allocated as 240mL.Flask is existed
TE-HER types CO2With 37.0 ± 0.5 DEG C of temperature, humidity 95%, CO in incubator (HIRASAWA company systems)2Concentration 5.0% enters one
Step 2 days (activation culture 2) of culture.After above-mentioned activation culture, the cell that OKT3 immobilization flasks make to be attached to bottom surface is patted
Peel off, obtained cell suspension.Obtained cell suspension is moved into new gas permeability culture bag (1000mL, Kohjin
Bio company systems 16087528) in, further (contain 1% human serum (Lymphotec company systems), 175U/mL with culture medium 2
rIL-2(Proleukin：Novartis company systems) AIM-V culture mediums：Invitrogen systems) to being cleaned in flask, will
In the cell suspension that the cleaning fluid is merged into the gas permeability culture bag, liquid measure is set to be 1000mL, in TE-HER types CO2
With 37.0 ± 0.5 DEG C of temperature, humidity 95%, CO in incubator (HIRASAWA company systems)2Concentration 5.0% is further cultivated 3 days
(amplification cultivation 1).After above-mentioned culture, 1000mL culture medium 2 is added in the culture bag newly used, it is sterilely connect
Be bonded in the culture bag cultivated in above-mentioned amplification cultivation 1, the content in two bags be sufficiently mixed, further after
It is continuous to be cultivated.The culture vessel be 37 DEG C, humidity 95%, CO2In the CO2gas incubator of concentration 5.0% further
Cultivate 4 days (amplification cultivation 2).
(4) analysis rolled into a ball to antibody Staining Lymphocyte
, will be flat using the date thawed to frozen cell as the 1st day in the process of (2) of the present embodiment 4~(3)
Bottom flask (225cm2) in culture terminate, to start at once before activation culture in OKT3 immobilization flasks as the 5th day,
Activation culture is terminated, started at once in gas permeability culture bag (1000mL, Kohjin Bio company systems 16087528)
As the 7th day before culture, it is engaged after the culture in gas permeability culture bag is terminated, with another gas permeability culture bag
The date for cultivating end afterwards, as the 14th day, (just lived in the 1st day (after just thawing), the 5th day (to activate at once before), the 7th day
After change) and the 14th day (to terminate at once before) with sterile manner gather culture in cell suspension a part, using with
(2) of embodiment 2 same method, for anti-cd 3 antibodies (BectonDickinson company systems 349201), anti-human
CD45RO antibody (BectonDickinson company systems 340438) and anti-human CD62L antibody (BectonDickinson
Pharmingen company systems 555544) analysis is implemented to the subgroup of the cell of acquisition.
As a result：
As a result it is shown in Table 7.It specify that the memory T cell that occupation rate rises in activation culture cell before freezen protective
Group still is able to maintain high presence ratio well after freeze-thaw during common culture and activation culture.Though
The right presence in the 7th day central type memory T cell is than showing reduction slightly, it has been observed that it is extensive by continuing to cultivate
It is multiple, be used as memory T cell group, although show in the presence of than slow reduction, but occupation rate has exceeded 80%.Can by the above results
To confirm, for the lymphocyte subgroup situation of the PBLC just collected from subject, note is resulted in
Recall activated lymphocyte (Fig. 2) of the T cell group to exist at high proportion." initial () " represent CD45RO-, CD62L+,
CD3+ T cells, " effect (Effector) " represents CD45RO-, CD62L-, CD3+ effector T cell, and CM is represented
CD45RO+, CD62L+, CD3+ central type memory T cell, EM represent that CD45RO+, CD62L-, CD3+ effect type memory T are thin
Born of the same parents, as CM+EM or " memory (memory) " represent CD45RO+, CD62L+/-, CD3+ memory T cell.
The change (%) of the lymphocyte surface antigen in the process of activation amplification is carried out after the chilled preservation of table 7
(5) stability of Cell Cryopreservation
Using the method same with above-described embodiment 4 (1), the cell of freezen protective will have been carried out simultaneously in being taken care of under liquid nitrogen
1 week, 1 month, 2 months, 4 months, 6 months, the stability to Cell Cryopreservation was studied (n=3).
The cell phenotype of cell amplification rate after cell number after defrosting, cell phenotype, defrosting and amplifying cells is carried out
Analysis, as a result finds, untill after freezen protective 6 months, cell number, survival rate and cell phenotype and solution after defrosting
Do not observe any difference in cell amplification rate and cell phenotype in culture processes after jelly.Therefore show, freezen protective
Cell is stable at least six month, and Cell Cryopreservation can be preserved stably for a long time.
Embodiment 5
The medical composition using memory cell as main component of the present invention is manufactured.The summary of manufacturing process such as Fig. 3 institutes
Show.
[manufacture of the invention using memory cell as the medical composition of main component]
1. the process of activation culture 1
The 225cm of anti-cd 3 antibodies (OKT3) in immobilization2Activation flask in inject to enter in the same manner as embodiment 1 (1)
Gone allotment 50mL cell suspending liquid, in 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ± 5.0%, CO2Concentration 5.0 ±
0.2% TE-HER types CO2Cultivated in incubator.When the cell suspending liquid amount of allotment is more than 100mL, burnt to 2 activation
The cell suspending liquid of each middle injection 50mL in bottle.Cell suspending liquid amount is more than 50mL but during less than 100mL, is injected
50mL.Start the 3rd day collection 0.01mL nutrient solution in culture, and measure suspension cell number (in-process inspection II).Suspension cell
Density is 4 × 105During individual/more than mL, add 50mL nutrient solutions 1 (nutrient solution is added).
In the in-process inspection II that culture starts the 3rd day, suspension cell density is not up to 4 × 105During individual/mL, followed by
Continuous culture 1 day simultaneously measures suspension cell number in next day (culture starts the 4th day)；Suspension cell density is 4 × 105Individual/more than mL
When, carry out nutrient solution and add process.
If suspension cell density reaches 4 × 10 when starting the 6th day to culture5Individual/mL, then carry out nutrient solution and add work
Sequence, in the case of being not up to, stops culture, is taken a blood sample again.
2. freezen protective process
Peel off the cell for being attached to flask bottom surface in the next day for having added nutrient solution, be equably suspended, sample 1mL
It is used as in-process inspection III samples., will by reclaiming cell from cell suspension within 5 minutes at room temperature with 1200rpm centrifugations
Cell is with 3 × 107Individual/pipe is freezed.Herein, cell is made with 3 × 107Individual/pipe is suspended in cell freezing and preserves liquid CP-1 (poles
Eastern pharmacy system) in, it is housed in cryopreservation tube, the pipe is inserted and frozen in cup, its snap frozen is made in -80 DEG C of refrigerators.Next day
Cryovial is taken out from -80 DEG C of freezers, is stored in liquid nitrogen.
3. defrosting treatment process
The test tube for having preserved Cell Cryopreservation is taken out from liquid nitrogen container, in 37 DEG C of dry type heating unit is heated to
Heating 4 minutes, thaws to frozen cell.Add what is thawed into the 15mL for the culture medium 1 for filling 10mL centrifuge tube
Cell liquid, is fully stirred by reverse mixing, is centrifuged 3 minutes with 1200rpm at room temperature.Supernatant is removed, makes cell point
Dissipate, then it is equably suspended with 40mL culture medium 1.Cell is sufficiently mixed, sampling 0.5mL is used as in-process inspection
IV sample.
Remaining cell suspending liquid is injected on the porous plate of flat 24 hole with 2mL/ holes, in 37.0 ± 0.5 DEG C of temperature, wet
Degree 95.0 ± 5.0%, CO2The CO of concentration 5.0 ± 0.2%2Culture 2 days in culture chamber.
4. expand culture processes
Cultivating the 3rd day by cell flat 225cm is moved to from 24 hole porous plates2In flask.It is porous with 1 pair of 24 hole of culture medium
Plate carries out cleaning to reclaim the cell of residual.Cell is suspended with culture medium 1, finally make into 90mL altogether, in temperature 37.0
± 0.5 DEG C, humidity 95.0 ± 5.0%, CO2The CO of concentration 5.0 ± 0.2%2Culture 2 days in incubator.
5. the process of activation culture 2
After culture 2 days, by flat 225cm2The activation that cell in flask moves to immobilization anti-cd 3 antibodies (OKT3) is burnt
In bottle.With culture medium 1 to flat 225cm2Flask is cleaned, and is reclaimed the cell of residual, is finally made Culture liquid measure be 250mL.
In 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ± 5.0%, CO2The CO of concentration 5.0 ± 0.2%2Culture 2 days in culture chamber.
6. the process of amplification cultivation 1
After culture 2 days, the cell for being attached to the bottom surface of activation flask is set to peel off and obtain cell suspending liquid.Gather 1mL's
Cell suspending liquid as in-process inspection V sample.Remaining 250mL cell suspending liquids are moved to and are filled with 750mL culture mediums 2
In the gas permeability culture bag of (with the addition of the culture medium of 1% human serum, it contains 175U/mL IL-2), in temperature 37.0 ± 0.5
DEG C, humidity 95.0 ± 5.0%, CO2The CO of concentration 5.0 ± 0.2%23 days (total Culture liquid measure about 1L) of culture in culture chamber.
7. the process of amplification cultivation 2
The culture bag for having cultivated 3 days is sterilely engaged with the gas permeability culture bag for the culture medium 2 for being filled with 1L, by two kinds
Nutrient solution is sufficiently mixed, in 37.0 ± 0.5 DEG C of temperature, humidity 95.0 ± 5.0%, CO2The CO of concentration 5.0 ± 0.2%2Incubator
4 days (total Culture liquid measure about 2L) of interior culture.
8. prepare and filling work procedure
After the beginning of amplification cultivation 2, in the 4th day (amount to the 14th~17 day) from 2 culture bags being joined together
A side each 250mL of nutrient solution is moved in 4 250mL centrifuge tube, at room temperature with 1,500rpm centrifuge 8 minutes.Remove
Supernatant, the nutrient solution in remaining 1 bag is moved in above-mentioned 250mL centrifuge tube.With bag originally under the same conditions
Centrifuged, remove supernatant.Disperse the cell in 4 250mL centrifuge tubes, the people that 0.1% is then with the addition of with 500mL is white
The physiological saline of albumen is suspended, and is collected in 2 250mL centrifuge tubes, in being centrifuged 8 minutes with 1,700rpm at 4 DEG C, to thin
Born of the same parents are cleaned.Repeat same operation, cleaning cell 2 times.Disperse the cell cleaned, be suspended in and be cooled to 4 DEG C
The 210mL human albumins that with the addition of 1% physiological saline in.Cell liquid of the 10mL through suspension is gathered as listing inspection
Sample.Remaining 200mL is filled to product filling bag, product has been made.
[analysis using memory cell as the medical composition of main component to the present invention]
Using the method same with (2) of embodiment 2, on anti-human CD45RO antibody (BectonDickinson companies
System) and anti-human CD62L antibody (BectonDickinsonPharmingen company systems), to being obtained by the method for the present invention
The activated lymphocyte and the subgroup of the activated lymphocyte made using conventional method obtained implements analysis (Fig. 4).
The method of the present invention is carried out according to the method described in embodiment 5.Culture is started in OKT immobilization flasks
When the monocyte (lymphocyte abundant component) (PBMC) isolated of being taken a blood sample from peripheral vascular carried out activation culture.
Reach 4 × 1054th day collection sample (+50) after/more than the mL activation culture, then adds 50mL culture medium 1, training
Support 1 day, collection sample (" freezing ") carries out freezen protective process afterwards.Gather sample (TW (solutions immediately after frozen cell defrosting
Freeze)) and culture processes are enlarged, activation culture 2 is carried out in OKT immobilization flasks thereafter.Gathered before activation culture 2 is faced
Sample (OKT), gathers sample (defrosting BP) immediately after.Thereafter amplification cultivation 2 is carried out in culture bag, lymphocyte is harvested,
The collection cell sample (defrosting H (Harvest)) before harvest is faced.
In conventional method, the monocyte that being taken a blood sample from peripheral vascular when starting is isolated will be cultivated with culture medium 1
Cell suspension is made in (the abundant component of lymphocyte) (PBMC), and the 50mL suspensions are carried out in OKT immobilization flasks
Activation culture.Reaching 4 × 1054th day collection sample (+50) after/more than the mL activation culture, then adds 50mL's
Culture medium 1, is cultivated 1 day.In the 5th day collection sample (+150), addition 150mL culture medium 1 was cultivated.Further, in
Next day collection sample (raw BP).Addition 100mL culture medium 1 is cultivated, finally into collection sample before culture (raw H).Need
It is noted that "+150 " and " freezing " sample is divided into sample obtained from being acquired immediately after freezing group and non-frozen group
Product, it is substantially the same.
The cell subsets of above-mentioned sample is studied, as a result in the conventional method not comprising freezing processing process
In, the ratio of memory T cell is 40% or so in the lymphocyte populations of acquisition, (Fig. 4 A) identical with PBMC degree, and uses this
During the method for invention, memory T cell accounts for about 75% (Fig. 4 B) in the lymphocyte populations of acquisition.Therefore, the method according to the invention,
Freezen protective is carried out by the lymphocyte to collection, convenience can be not only improved, can also obtain with adoptive immunity
The lymphocyte populations that the memory T cell of active ingredient is main component are used as in therapy etc..
Osumi et al. is conceived to illustrate the mechanism of action of the activated lymphocyte used in adoptive immunotherapy
The function for the activated lymphocyte that memory T cell subgroup is obtained to activation amplification cultivation is analyzed.As a result, confirming to live
Change the effect type memory T cell included in lymphocyte and show cell inhibitory activity to cancer cell at 24 hours, and
Central type memory T cell is activated after tumour antigen is identified, and is divided into effect type memory T cell, is presented at 48 hours
Go out cell inhibitory activity.
Hereinafter, the effect of central type memory T cell and effect type memory T cell to tumour cell is provided.It is anti-human to whether there is
CD45RO antibody (BectonDickinson company systems) and anti-human CD62L antibody (BectonDickinson
Pharmingen company systems) it is index, with flow cytometer (BectonDickinson company systems；FACS Calibur) will
The Autologous lymphocyte (KC-362) of activation is divided into central type memory T cell (CM) and effect type memory T cell (EM) group, and (CM is pure
Spend 99.6%, EM purity 98.5%).Then, by isolated each cell mass and the KT-362 (tumour cells from patient
Strain) co-cultured, cell disorders activity is analyzed (table 8).
Table 8
During KT-362 independent (control group), compared with cell number when starting with culture, cell number increased at 24 hours
5.3 times are increased to 2.5 times, at 48 hours.On the other hand, when being co-cultured with CM, pressed down it was observed that breeding at 24 hours
0.9 times is made as, was reduced at 48 hours to 0.3 times.After incubation 48 hours when reclaim lymphocyte, use flow cytometer
(BectonDickinson company systems；FACS Calibur) analyzed, as a result find that CM antigenic type becomes EM.
It is thin at 24 hours compared with cell number when starting with the culture of control group in KT-362 and EM co-cultivation
Born of the same parents' number is reduced to 0.2 times, and living cells is not observed at 48 hours.
Therefore, CM and EM are respectively provided with cell inhibitory activity, although wherein CM existence times in the presentation of inhibitory activity are poor,
But because its life-span is much longer compared with EM, it is believed that its supply source in the long-term interior stabilization as EM, from this point of view,
Show that the composition containing the lymphocyte populations for selectively having expanded CM can turn into effective therapeutic agent.
There is problems with the activated lymphocyte group obtained using conventional method：Can only be in PBMC collection post activation trainings
The high activated lymphocyte of central type memory T cell occupation rate is obtained in a period of supporting 3~7 days or so limited, and herein
Period cell number not yet fully increases, and after cell number fully increases, central type memory T cell occupation rate will be reduced to
Less than 20% (non-patent literature 3).On the other hand, according to the method for the present application, cell number can fully be increased, and
Result in the activated lymphocyte group of the invention that the CM compared with EM accounts for absolute majority, although for cell inhibitory activity compared with
EM has certain delay in terms of active presentation, but think by just activity itself it is inferior unlike EM it is this bestow, energy
The high EM of cell inhibitory activity supply steady in a long-term is enough realized, so as to realize effective therapeutic effect.It is additionally, since comprising thin
Born of the same parents' freezing stage, the freezing period are not particularly limited, therefore can also be fast for long-term keeping and eager demand
The high activated lymphocyte group of memory T cell (particularly central type memory T cell) occupation rate is prepared fastly, from this point of view,
Convenience is very high.
Industrial applicability
The present invention can suitably be used in therapeutic agent field comprising activated lymphocyte etc..
Claims (6)
1. using memory T cell as the manufacture method of the lymphocyte populations of main component, the manufacture method using include successively with
Lower process (a)~(c) high proliferation cultivation is carried out,
Process (a), 4.0 × 10 are adjusted to by the lymphocyte of collection5Individual/more than mL, 8.0 × 105Individual/below mL inoculation is thin
Born of the same parents' density, IL-2 and immobilization anti-cd 3 antibodies and suitable for cultivate lymphocyte nutrient solution in the presence of carry out activation training
Support；
Process (b), is 4 × 10 based on the lymphocyte number being suspended in nutrient solution5Individual cell/more than mL, detects memory T thin
The occupation rate of born of the same parents group rises, and the lymphocyte of process (a) is carried out into freezen protective；
Process (c), is thawed, activation culture to the lymphocyte being frozen.
2. manufacture method as claimed in claim 1, it is characterised in that process (c) is completed within after defrosting 2 weeks.
3. manufacture method as claimed in claim 1 or 2, it is characterised in that using memory T cell as the lymphocyte of main component
Group is that the occupation rate of memory T cell in lymphocyte populations is more than 75% lymphocyte populations.
4. manufacture method as claimed in claim 1 or 2, it is characterised in that memory T cell with account for the 70% of lymphocyte populations with
On ratio contain central type memory T cell.
5. manufacture method as claimed in claim 1 or 2, it is characterised in that separated lymphocyte comes from human peripheral.
6. manufacture method as claimed in claim 1 or 2, it is characterised in that lymphocyte populations are for bestowing to the third party
Cell mass.
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|---|---|---|---|
| JP2012088926A JP6142142B2 (en) | 2012-04-10 | 2012-04-10 | Method for producing lymphocyte cell group mainly comprising memory T cells |
| JP2012-088926 | 2012-04-10 | ||
| PCT/JP2013/002405 WO2013153800A1 (en) | 2012-04-10 | 2013-04-09 | Method for producing lymphocyte cell group consisting mainly of memory t cells |
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| CN104204195B true CN104204195B (en) | 2017-07-18 |
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| JP (1) | JP6142142B2 (en) |
| KR (1) | KR101521484B1 (en) |
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| JP6424447B2 (en) | 2014-03-28 | 2018-11-21 | 東洋製罐グループホールディングス株式会社 | Cell culture method and cell culture system |
| CN106796237B (en) * | 2014-07-22 | 2018-12-04 | 株式会社癌症免疫研究所 | The detection device of peripheral circulation cancer cell |
| EP3252140B1 (en) | 2015-01-30 | 2022-03-02 | Toyo Seikan Group Holdings, Ltd. | Cell culturing method and cell culturing device |
| CN113061577A (en) * | 2017-09-05 | 2021-07-02 | 四川新生命干细胞科技股份有限公司 | Isolated culture method of high-purity NK cells |
| CN111601883A (en) * | 2017-11-17 | 2020-08-28 | 艾欧凡斯生物治疗公司 | TIL amplification from Fine needle aspirates and Small biopsies |
| JP2021533759A (en) * | 2018-08-10 | 2021-12-09 | ユーティレックス カンパニー リミテッド | Methods for preparing and cryopreserving cancer antigen-specific CD8 + T cells |
| CN109628396B (en) * | 2019-01-24 | 2021-03-26 | 清华大学 | Application of memory lymphocyte population in liver cancer treatment |
| CN113005081B (en) * | 2019-12-20 | 2023-12-19 | 苏州依科赛生物科技股份有限公司 | Culture method for amplifying stem cell-like memory T cells |
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| CN1363681A (en) * | 2000-12-04 | 2002-08-14 | 胡曼泰克有限公司 | Activated lymphocyte from umbilical cord blood, preparations containing it as main content and method therefor |
| CN1230524C (en) * | 2000-12-04 | 2005-12-07 | 株式会社淋巴技术 | Cell-preservation liquid and method of preserving cells using the liquid |
| EP1066833B1 (en) * | 1999-07-08 | 2008-10-01 | Lymphotec Inc. | Use of cryopreserved activated lymphocytes as a medicament |
| JP4389039B2 (en) * | 1998-04-09 | 2009-12-24 | 株式会社リンフォテック | PHARMACEUTICAL COMPOSITION CONTAINING ALLOGENIC ACTIVATED CD4-POSITIVE CELLS AS MAIN COMPONENTS, PROCESS FOR PRODUCING THE SAME AND KIT FOR PREPARING THE PHARMACEUTICAL COMPOSITION |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4385158B2 (en) * | 2000-12-04 | 2009-12-16 | 株式会社リンフォテック | Cell preservation solution and cell preservation method using the preservation solution |
| JP5977238B2 (en) * | 2010-09-08 | 2016-08-24 | イェダ リサーチ アンド デベロップメント カンパニー リミテッド | Use of anti-third party central memory T cells for anti-leukemia / lymphoma treatment |
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2012
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP4389039B2 (en) * | 1998-04-09 | 2009-12-24 | 株式会社リンフォテック | PHARMACEUTICAL COMPOSITION CONTAINING ALLOGENIC ACTIVATED CD4-POSITIVE CELLS AS MAIN COMPONENTS, PROCESS FOR PRODUCING THE SAME AND KIT FOR PREPARING THE PHARMACEUTICAL COMPOSITION |
| EP1066833B1 (en) * | 1999-07-08 | 2008-10-01 | Lymphotec Inc. | Use of cryopreserved activated lymphocytes as a medicament |
| CN1363681A (en) * | 2000-12-04 | 2002-08-14 | 胡曼泰克有限公司 | Activated lymphocyte from umbilical cord blood, preparations containing it as main content and method therefor |
| CN1230524C (en) * | 2000-12-04 | 2005-12-07 | 株式会社淋巴技术 | Cell-preservation liquid and method of preserving cells using the liquid |
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| A feasible method for expansion of peripheral blood lymphocytes by culture with immobilized anti-cd3 monoclonal antibody and interleukin-2 use in a doptive immunotherapy of cancer patients;T Sekine 等;《BIOMED PHARMACOTHER》;19930331;第47卷(第2-3期);73-78 * |
| Ex vivo expansion of umbilical cord blood for transplantation;S.S.Tung 等;《Best Practice & Research Clinical Haematology》;20100630;第23卷(第2期);245-257 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101521484B1 (en) | 2015-06-05 |
| WO2013153800A1 (en) | 2013-10-17 |
| CN104204195A (en) | 2014-12-10 |
| HK1205181A1 (en) | 2015-12-11 |
| JP2013215141A (en) | 2013-10-24 |
| KR20140145585A (en) | 2014-12-23 |
| JP6142142B2 (en) | 2017-06-07 |
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