CN106701677A - In-vitro expansion culture method of NK cells - Google Patents
In-vitro expansion culture method of NK cells Download PDFInfo
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Abstract
The invention relates to an in-vitro expansion culture method of NK cells. The culture method comprises the following steps that the NK cells are acquired; the NK cells are subjected to in-vitro expansion culture in a culture medium containing an insulin-like growth factor-1. The insulin-like growth factor-1 replaces feeder cells for carrying out expansion culture on the NK cells, so that the steps of NK cell expansion culture are simplified; the cell viability, the multiplication rate and the killing activity of the NK cells can be improved through the insulin-like growth factor-1, the insulin-like growth factor-1 can stimulate the NK cells to secrete the endogenic insulin-like growth factor-1 to further improve the killing activity of the NK cells, and therefore the defects that in the prior art, due to the fact that the feeder cells are used for carrying out multiplication culture on the NK cells, the NK cell multiplication rate is low, and the killing activity is poor are overcome.
Description
Technical field
The present invention relates to bioengineering and biomedicine field, more particularly to a kind of external expansion of NK cells
Increase cultural method.
Background technology
NK (Natural killer cells, NK cell), as arranged side by side with T cell, B cell
A major class lymphocyte population, be the important member of natural immune system.NK cells are congenital except participating in
Outside property immune response, important regulating and controlling effect is also played in the immunological regulation of adaptive immunity.NK is thin
The target cell of born of the same parents mainly have some tumour cells (including part cell line), virus infected cell, some from
Body histocyte (such as haemocyte), parasite, therefore, NK cells are antitumor, anti-infective bodies
Important immune factor, also assist in the IIth type hypersensitivity and graft-versus-host reaction.To sum up, NK is thin
The key of promotion clinical frequent application when the in vitro amplification and activation of born of the same parents, and the amplification side of NK cells at present
Method separates NK precursors or CD56 and using steps such as feeder cells before being mainly included in culture, not only needs
Want feeder cells, operation cumbersome, and the expanding effect of NK cells is not obvious, and the NK for being obtained
Cell killing activity is poor.
The content of the invention
The technical problems to be solved by the invention are that it is thin to there is NK for prior art amplification NK cells
The defect such as born of the same parents' amplification rate is low and killing activity is poor, there is provided one kind can improve NK cell amplification rates and killing
The amplification in vitro method of the NK cells of activity.
The technical solution adopted for the present invention to solve the technical problems is：A kind of the external of NK cells is provided
Amplification cultivation method, the cultural method is comprised the following steps：
Obtain NK cells；
Amplification in vitro training is carried out to the NK cells in the culture medium containing insulin-like growth factor-Ⅱ l
Support.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, in the culture medium, pancreas islet
The concentration of plain like growth factor -1 is 50-150ng/ml.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, the process of NK cells is obtained,
Comprise the following steps：
Obtain CD34+Candidate stem cell enters in the stem cell media containing hematopoietic stem cell growth factor
Assassinate to swash and be divided into NK cells.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, the hematopoietic stem cell growth
The factor includes stem cell factor, FLT3L and interleukin-15.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, in the stem cell media,
The concentration of stem cell factor is 10-50ng/ml, the concentration of FLT3L is
20-80ng/ml, and the concentration of interleukin-15 is 20-80ng/mI.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, body is carried out to the NK cells
The step of outer amplification cultivation is：The candidate stem cell that contains is added to give birth to the insulin-like growth factor-Ⅱ l
Culture 20-50 days is carried out in the stem cell media of the factor long.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, the CD34+Hematopoietic Stem is thin
The acquisition process of born of the same parents, comprises the following steps：
Mononuclearcell is isolated from haemocyte, CD34 is gone out through magnetic bead sorting+Candidate stem cell.
In the amplification in vitro cultural method of the NK cells that the present invention is provided, the blood cell collection is outside
All blood, Cord blood, decidua tissue and/or lymph node.
Implement the NK cell expansion ex vivo cultural methods that the present invention is provided, following beneficial effect can be reached：
The present invention carries out amplification cultivation instead of feeder cells by using insulin-like growth factor-Ⅱ l to NK cells,
The step of not only simplify NK cell amplification cultivations, and can be improved by insulin-like growth factor-Ⅱ l
The cytoactive of NK cells and the killing activity of amplification rate and NK cells, while insulin-like growth factor
Son-l can also stimulate NK cells to secrete endogenic insulin-like growth factor-Ⅱ l, further to improve NK
The killing activity of cell, so as to solve in the prior art, is expanded NK cells by feeder cells
Increase culture and there is the defects such as NK cell amplification rates are low, killing activity is poor.
Specific embodiment
To solve the presence of amplification by way of feeder cells carry out NK cell amplification cultivations in the prior art
The defects such as rate is low, NK cell killing activity differences, innovative point of the invention is to provide a kind of NK cells
Amplification cultivation mode, enables that NK cells are constantly in increasing by using insulin-like growth factor-i
State is grown, and its cytoactive and functional characteristic can be improved, improve killing activity of NK cells etc.,
And without being the amplification that can reach NK cells using feeder cells, used in the prior art so as to solve
Complex operation that feeder cells culture NK cells are present, NK cell amplification rates are low and killing activity is poor etc. lacks
Fall into.
Specifically, the amplification in vitro cultural method of the NK cells that the present invention is provided, comprises the following steps：
S1, acquisition NK cells；
S2, NK cells are inoculated in the culture medium containing insulin-like growth factor-i and are expanded in vitro
Increase culture.
Further, in step sl, NK cells may be from peripheral blood, Cord blood, decidua tissue
And/or lymph node.NK cell surface expression CD56 antigens, CD3 antigens are not expressed, according to CD56
The different expression of antigen, NK cells can be divided into CD56dimCD16brightAnd CD56brightCD16dim/negTwo
Big subgroup；CD56brightCD16dim/negNK cells are then primarily present in lymph node and decidua tissue,
With secrete cytokines such as interferon-γ as major function, killing ability is weaker；And CD56dimCD16bright
NK cells are then primarily present in peripheral blood, there is abundant intracellular perforin, and killing ability is stronger, therefore,
In the present invention, it is preferential using the acquisition NK cells from peripheral blood.
NK cells are mainly by CD34+Candidate stem cell divides under the stimulation of hematopoietic stem cell growth factor
It is ripe NK cells to change development, and the stage of development of NK cells is：From CD34+Candidate stem cell first divides
Turn to common lymphoid sample precursor cells, common lymphoid sample precursor cells regeneration NK precursors, before NK
Somatocyte development is immature NK cells, and finally, immature NK cells obtain reactivity and inhibition is received
Body, and it is ripe NK cells to obtain functional development.
Further, the process of NK cells is obtained, is comprised the following steps：Obtain CD34+Hematopoietic Stem is thin
Born of the same parents are cultivated in the stem cell media containing hematopoietic stem cell growth factor.
Wherein, for by CD34+Hematopoietic stem cell growth of the candidate stem cell differentiation and development into NK cells
The factor, including stem cell factor, FLT3L and interleukin-15；And preferably,
In stem cell media, the concentration of stem cell factor is 10-50ng/ml, FMS sample EGFR-TK 3
The concentration of part is 20-80ng/ml, the concentration of interleukin-15 is 20-80ng/mL.
Stem cell factor (stem cell factor, SCF) is a kind of by grappling and to be expressed in all Hematopoietic Stems thin
The tyrosine kinase receptors c-Kit of cellular surface and the factor that plays a role, it is to avoid c-Kit expression defect causes hematopoiesis
The reduction of expansion of stem cells quantity.
FLT3L (Flt-3L) acts on CD34+Candidate stem cell, by cell
Surface is combined with TKR and is played Hematopoietic Regulation, has obvious rush to the amplification in vitro of candidate stem cell
Enter effect.SCF and Flt-3L belongs to tyrosine kinase receptor TKR families, with amplification primitive hematopoietic
The synergy of stem cell, is combined by with specificity T KR, to intracellular delivery signal, starts early stage
The division of candidate stem cell and propagation, make cell walk out the G0 phases, start amplification, suppress apoptosis.
Interleukin-15 can not only promote propagation and the differentiation of candidate stem cell, and NK can be promoted thin
The expression of born of the same parents NKG2D this Activating receptor, strengthens NK cell killing related genes such as perforin
Expression, so as to strengthen the killing ability of NK cells.
Under the stimulation of these growth factors, candidate stem cell quantity increase, and candidate stem cell with
Break up its phenotype also to change, the mesoderm growing early stage stage, the IL-2/IL-15R- β cell factors on stem cell
Raising occurs in the expression of acceptor, i.e. CD122, and stem cell is divided into NK precursors therewith, before NK
It is the ability of maturation NK cells that body cell has orientation development, but NK precursor cell surfaces do not express CD56
Antigen；Meanwhile, NK precursors are that IL-2/IL-15 receptor βs are raised because of expression CD122 molecules, institute
Very sensitive with the stimulation enhancing to interleukin-15, interleukin-15 can promote the development of NK cells,
Interleukin-15 is mostly come from bone marrow microenvironment；Additionally, IL-15 can expand NK cell precursors
And it is divided into the ripe NK cells of the various Inhibitory receptors of expression and Activating receptor.
Further, also include obtaining CD34 in step S1+The process of candidate stem cell, and CD34+Make
Hemocytoblast is then after isolating mononuclearcell from peripheral blood haemocyte, to go out through magnetic bead sorting.
In step S2, culture medium is stem cell media, can be with culture CD34+Candidate stem cell is adopted
Stem cell media is consistent；Preferably, culture medium is SCGM (GMP Serum-free Stem
Cell Growth Medium) culture medium, purchased from German CellGenix companies.Insulin-like growth factor
- 1 (insulin-like growth factors-1, abbreviation IGF-1) of son can promote the development and amplification of NK cells,
And the killing activity of NK cells can be promoted, and NK cells can be promoted to secrete IGF-1 in itself, this
Plant endogenic IGF-1 most important to the killing ability of NK cells；Preferably, IGF-1 in culture medium
Concentration be 50-150ng/ml.
Further, in the present invention, more simplified process steps are：
CD34 is isolated from peripheral blood+Candidate stem cell, is then inoculated in and is added with candidate stem cell life
Culture 20-50 days is carried out in the stem cell media of the factor long and IGF-1, you can harvest the NK after amplification
Cell；Wherein, hematopoietic stem cell growth factor includes stem cell factor, FLT-3 parts and interleukin-15.
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments,
The present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to
The present invention is explained, is not intended to limit the present invention.
Specific embodiment is：
Embodiment 1
The amplification in vitro cultural method of the NK cells that the present invention is provided, comprises the following steps：
S1a, acquisition NK cells；
The separation of S11a, mononuclearcell；
Gather 100ml human peripherals blood sample, or 80mL umbilical cord bloods blood sample, with 1-2 times of body
Long-pending PBS solution dilution blood sample, after fully mixing, obtains PBS blood sample mixed liquors, then in each 50ml
The Ficoll separating liquids of 15ml are first added in centrifuge tube, the density of Ficoll separating liquids is 1.077g/mL；Again
The PBS blood sample mixed liquors of 20-30ml are slowly added to, room temperature centrifugation, centrifuge lifting speed is adjusted to 0,
In 20 DEG C, 400g, 30min is centrifuged, intermediate layer is mononuclearcell；Carefully drawn with pasteur pipet,
Afterwards centrifuge tube is filled it up with 1 × PBS, abundant centrifuge washing mononuclearcell (400g is centrifuged l0min),
Re-suspended cell, it is standby.
S12a、CD34+The sorting of candidate stem cell；
The mononuclearcell that will be obtained in step S11a after Trypan Blue living cell counting being adjusted to 2 × 108
The cell suspension of individual/m1, the ratio of 100 μ L antibody is added in every milliliter of cell suspension, adds CD34
Mixtures of antibodies, fully mixing is blown and beaten with pipettor up and down, is incubated at room temperature 15 minutes；Then every milliliter is pressed
Cell suspension adds the ratio of 50 μ L magnetic beads, adds CD34+Magnetic bead fully blows and beats mixing, incubation at room temperature 10
After minute, cell is transferred to polystyrene tube (12 × 75rnm), plus PBS cleaning solutions (containing 2%FBS,
0.01%EDTA) to 2.5ml, in vitro gently blown and beaten 2~3 times up and down with pipettor, mix cell.
Again by test tube insertion magnetic pole, five minutes are stood；Then magnetic pole is picked up together with test tube, continuously to delay
Magnetic pole to inversion state is toppled in slow action, pours out supernatant fraction.The cell of magnetic mark is due to magnetic pole magnetic
The attraction of field, still remains in test tube.Keep magnetic pole and test tube to be inverted 2~3 seconds, then make test tube mouthful extensive
Multiple upward position.Test tube is taken out from magnetic pole, 2.5ml cleaning solutions are added, is gently blown and beaten with pipettor thin
Born of the same parents' suspension 2~3 times, mixes cell, then by tube back magnetic pole, stand five minutes.Repeated washing 4
After secondary, that retain in test tube is CD34+Candidate stem cell, it is standby.
S2a, by CD34+Candidate stem cell is inoculated in dry containing hematopoietic stem cell growth factor and IGF-1
Amplification cultivation is carried out in cell culture medium；
Adjustment CD34+The cell suspension density of candidate stem cell is 1 × 105Individual/ml, and be inoculated in containing
In 24 orifice plates of SCGM stem cell medias (being purchased from CellGenix companies of Germany), done before inoculation
CD34 before flow cytometer showed detection inoculation+Candidate stem cell content, and ensure CD34+Candidate stem cell is 95%
More than；Add stem cell factor, FLT-3L and interleukin-15 and IGF-1 respectively again, and cause dry thin
In born of the same parents' culture medium, the concentration of stem cell factor is 50ng/ml and Bai Jie for the concentration of 30ng/ml, FLT-3L
The concentration of element -15 is 100ng/mL for the concentration of 50ng/mL and IGF-1；In 37 DEG C, 5%CO2Incubate
Case culture 28 days.
Embodiment 2
Difference with embodiment 1 is, the hematopoietic stem cell growth factor added in the embodiment
And concentration of the IGF-1 in stem cell media is respectively：The concentration of stem cell factor be 10ng/ml,
The concentration of FLT-3L is 20ng/ml and the concentration of interleukin-15 is for the concentration of 20ng/mL and IGF-1 is
50ng/mL。
Embodiment 3
Difference with embodiment 1 is, the hematopoietic stem cell growth factor added in the embodiment
And concentration of the IGF-1 in stem cell media is respectively：The concentration of stem cell factor be 50ng/ml,
The concentration of FLT-3L is 80ng/ml and the concentration of interleukin-15 is for the concentration of 80ng/mL and IGF-1 is
150ng/mL。
Further to verify the remarkable result of the NK cell expansion ex vivo cultural methods that the present invention is provided, lead to
Crossing following experiment carries out detection checking.
Detection object：
Control group：It is that the group uses keratinocytes media with the difference of the embodiment of the present invention 1
The cell that NK cell amplification cultivations are obtained is carried out with proleulzin and feeder cells；
Detection group 1-3：The cell that respectively embodiment of the present invention 1-3 is obtained.
1st, the flow cytometry analysis of NK cells
The NK cells after amplification are counted using Becton Dickinson types flow cytometers, is detected
Result such as table 1 below.
Table 1
As can be seen from the above results, with the increase of cultivated days, cell quantity in detection group 1-3 by
It is cumulative to add, and far above the cell quantity in control group；It follows that the NK provided by the present invention
The NK cell quantities that the amplification in vitro cultural method of cell is obtained are more, improve the amplification of NK cells
Rate.
2nd, the detection of NK cell killing activities
Collect the K562 cells (HEL's K562 cells, buy) of exponential phase, weight
It is suspended from the RPMI1640 culture mediums containing 10%FCS of 0.5ml, by 100 μ Ci/L × 106Cell adds
Enter 51Cr.In 37 ° DEG C, 5%CO22h is incubated in incubator, is shaken up once every 15min, marked
Washed 3 times after finishing, adjustment cell concentration to 1 × 105/ mL is added in the orifice plate of round bottom 96,100 μ L/ holes,
As the target cell in killing experiments.By 10:1、5:1 and 2.5:NKs of the 1 equivalent IE than addition respective amount
Cell, in 37 DEG C, 5%CO24h is incubated in incubator, 100 μ L of supernatant are collected per hole, survey gamma-rays
Cpm values, calculate killing rate as follows：Killing rate (%)=(experimental group cpm values-Spontaneous release cpm
Value)/(maximum release cpm values-Spontaneous release cpm values) × l00%, detection data see the table below 2：
Table 2
| Effect target compares E/T | Control group | Detection group 1 | Detection group 2 | Detection group 3 |
| 10∶1 | 27.7±1.9 | 50.2±2.9 | 48.5±2.1 | 52.5±2.7 |
| 5∶1 | 15.4±3.1 | 29.6±3.5 | 27.4±3.7 | 31.9±3.2 |
| 2.5∶1 | 9.3±1.8 | 17.3±3.1 | 15.7±3.6 | 19.6±3.3 |
Testing result：From data in table 2, the cell killing rate of detection group 1-3 is all higher than control group,
Thus illustrate, the amplification cultivation method of the NK cells that the present invention is provided improves the killing activity of NK cells.
In sum, in the amplification in vitro cultural method of the NK cells that the present invention is provided, using insulin
Like growth factor-l carries out amplification cultivation NK cells, can not only promote the development and amplification of NK cells,
The cytoactive and amplification rate of NK cells are improved, but also NK cells itself can be promoted to secrete IGF-1,
To improve the killing activity of NK cells, the clinical practice for NK cells has great importance, and solves
In the prior art, the operating process that exists by feeder cells amplification cultivation NK cells is complicated, NK is thin
Born of the same parents' amplification rate is low, killing activity difference the problems such as.
It is described above in association with embodiments of the invention, but the invention is not limited in above-mentioned tool
Body implementation method, above-mentioned specific embodiment is only schematical, rather than restricted, ability
The those of ordinary skill in domain is not departing from present inventive concept and claim is protected under enlightenment of the invention
Under the ambit of shield, many forms can be also made, these are belonged within protection of the invention.
Claims (8)
1. a kind of amplification in vitro cultural method of NK cells, it is characterised in that：The cultural method includes
Following steps：
Obtain NK cells；
Amplification in vitro training is carried out to the NK cells in the culture medium containing insulin-like growth factor-Ⅱ l
Support.
2. the amplification in vitro cultural method of NK cells according to claim 1, it is characterised in that
In the culture medium, the concentration of insulin-like growth factor-i is 50-150ng/ml.
3. the amplification in vitro cultural method of NK cells according to claim 1, it is characterised in that
The process of NK cells is obtained, is comprised the following steps：
Obtain CD34+Candidate stem cell enters in the stem cell media containing hematopoietic stem cell growth factor
Assassinate to swash and be divided into NK cells.
4. the amplification in vitro cultural method of NK cells according to claim 3, it is characterised in that
The hematopoietic stem cell growth factor includes stem cell factor, FLT3L and Bai Jie
Element -15.
5. the amplification in vitro cultural method of NK cells according to claim 4, it is characterised in that
In the stem cell media, the concentration of stem cell factor is 10-50ng/ml, FMS sample EGFR-TK
The concentration of 3 parts is 20-80ng/ml, and the concentration of interleukin-15 is 20-80ng/mI.
6. the amplification in vitro cultural method of NK cells according to claim 3, it is characterised in that
The step of carrying out amplification in vitro culture to the NK cells be：The insulin-like growth factor-Ⅱ l is added
Culture 20-50 days is carried out in the stem cell media containing hematopoietic stem cell growth factor.
7. the amplification in vitro cultural method of NK cells according to claim 3, it is characterised in that
The CD34+The acquisition process of candidate stem cell, comprises the following steps：
Mononuclearcell is isolated from haemocyte, CD34 is gone out through magnetic bead sorting+Candidate stem cell.
8. the amplification in vitro cultural method of NK cells according to claim 6, it is characterised in that
The blood cell collection is from peripheral blood, Cord blood, decidua tissue and/or lymph node.
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