U.S. patent application number 15/486562 was filed with the patent office on 2017-10-19 for use of tumor dissociation reagent in flow cytometry.
The applicant listed for this patent is WuXi AppTec (Suzhou) Co. Ltd.. Invention is credited to Qunsheng JI, Yan LIU, Ning ZHANG, Qiyao ZHANG.
Application Number | 20170299491 15/486562 |
Document ID | / |
Family ID | 56459876 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170299491 |
Kind Code |
A1 |
ZHANG; Ning ; et
al. |
October 19, 2017 |
USE OF TUMOR DISSOCIATION REAGENT IN FLOW CYTOMETRY
Abstract
The present disclosure relates to a dissociation reagent for
tumor tissues. The dissociation reagent does not contain
collagenase or trypsin but further contains hyaluronidase or a
mixture of hyaluronidase and DNase I. The present disclosure also
relates to use of the dissociation reagent in dispersing tumor
tissues and detecting expression level of molecular markers on cell
surface by flow cytometry. The dissociation reagent of the present
disclosure does not cause degradation of molecular markers on cell
surface such as CD8, PD-1, Tim-3, Lag-3 and the like, thus does not
affect downstream assays.
Inventors: |
ZHANG; Ning; (Suzhou,
CN) ; ZHANG; Qiyao; (Suzhou, CN) ; LIU;
Yan; (Suzhou, CN) ; JI; Qunsheng; (Suzhou,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WuXi AppTec (Suzhou) Co. Ltd. |
Suzhou |
|
CN |
|
|
Family ID: |
56459876 |
Appl. No.: |
15/486562 |
Filed: |
April 13, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/483 20130101;
G01N 33/574 20130101; G01N 15/00 20130101; G01N 33/535 20130101;
G01N 15/1459 20130101; G01N 1/4044 20130101; G01N 2015/1006
20130101; G01N 15/14 20130101; G01N 33/56966 20130101; G01N
33/57484 20130101; G01N 33/48 20130101 |
International
Class: |
G01N 15/14 20060101
G01N015/14; G01N 33/569 20060101 G01N033/569; G01N 33/483 20060101
G01N033/483; G01N 33/574 20060101 G01N033/574; G01N 33/535 20060101
G01N033/535 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 2016 |
CN |
201610235031.4 |
Claims
1. A tumor dissociation reagent which does not comprise collagenase
but comprises hyaluronidase, wherein the tumor dissociation reagent
does not degrade or partially degrade membrane surface
receptor.
2. The tumor dissociation reagent of claim 1, which further does
not comprise trypsin.
3. The tumor dissociation reagent of claim 1, which further
comprises DNase I.
4. The tumor dissociation reagent of claim 1, wherein the membrane
surface receptor is a checkpoint receptor.
5. The tumor dissociation reagent of claim 1, wherein the membrane
surface receptor comprises CD8, PD-1, PD-L1, TIM-3 or LAG-3
protein.
6. A method for detecting protein expression level of an
immunological checkpoint marker in tumor tissue, comprising
applying the tumor dissociation reagent of claim 1 to the tumor
tissue.
7. The method of claim 6, wherein the tumor dissociation reagent
further comprises DNase I.
8. The method of claim 6, wherein the tumor tissue includes tumor
infiltrating immune cell.
9. The method of claim 6, wherein the protein expression in tumor
tissue is detected by flow cytometry.
10. The method of claim 6, wherein the protein is membrane surface
receptor.
11. The method of claim 10, wherein the membrane surface receptor
comprises CD8, PD-1, PD-L1, TIM-3 or LAG-3 protein.
12. A kit for tumor dissociation comprising the tumor dissociation
reagent of claim 1.
13. The kit of claim 12, wherein the tumor dissociation reagent
further comprises DNase I.
14. A method of detecting protein expression in tumor tissue by
flow cytometry comprising applying the kit of claim 12 to the tumor
tissue.
15. A method of preventing degradation of an immunological
checkpoint marker in tumor tissue, comprising treating cells with
the tumor dissociation agent of claim 1.
16. The method of claim 15, wherein the tumor dissociation reagent
further comprises DNase I.
17. The method of claim 15, comprising dissolving the tumor tissue
with the tumor dissociation reagent, and detecting the
immunological checkpoint marker by flow cytometry.
18. The method of claim 17, wherein the tumor tissue includes tumor
infiltrating immune cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to Chinese Application No.
201610235031.4 filed on Apr. 15, 2016, which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates to a tumor dissociation
reagent useful in flow cytometry for dispersing a clinical solid
tumor tissue into a single cell suspension and for protecting the
surface marker from being degraded.
BACKGROUND
[0003] In the analysis of the cell biological characteristics of
tumor tissue (such as the detection of cell epitope, etc.),
preparation in tissue blocks of the sample into a single cell
suspension is required, so as to obtain high-yield cells, and
integrity of cells and epitopes is intact and can be used directly
in downstream experiments. Only when various cell components of the
sample tissue are in a single cell state, a variety of detection
and analysis of cell effects can be effectively carried out, while
the quality of cell suspension is closely related to digestive
fluid formulations and digestion methods. The preparation methods
of single cell suspension commonly used are chemical, enzymatic and
physical methods.
[0004] The cell-to-cell linkages such as collagenase, elastic
fiber, mucopolysaccharide, tight junction proteins and the like are
mainly destructed for cell dispersion in enzymatic method. Trypsin,
collagenase, hyaluronidase and the like are the commonly used
enzymes. In prior art, it is stated that a variety of tissues
including tumor tissue, skeletal muscle, spleen, lung, nerve
tissue, epidermis, lamina propria, mouse heart, neonatal neurons,
embryoid bodies and the like may be treated with commercial
dissociation kits such as the Miltenyi Tissue Dissociation Kit; and
according to the difference in the sensitivity of the cell surface
antigen to enzymatic digestion, different enzyme reaction systems
are designed for preventing the cell surface antigen destruction,
so as not to affect downstream experiments. In fact, there are some
molecular markers on cell surface that are degraded or partially
degraded in practices, making it impossible to detect
accurately.
[0005] In fact, some commonly used components of the enzymatic
reagent will affect the detection of cell surface molecular
markers. For example, Trypsin has a strong ability to disperse
cells with short action time. The use of trypsin in preparation of
single cell suspension results in high yield, but the conditions of
action required by trypsin are complex. In addition, Trypsin may
damage the cell surface antigen and even cells. For example,
trypsin-treated mouse thymus cell surface receptors CD4 and CD8 are
digested by trypsin digestion (Thomas Barthlott, Rebecca J. Wright
and Brigitta Stockinger. J Immunol, 1998. 161:3992-3999).
Therefore, trypsin is suitable for detection of intracellular
antigens but not for cell surface antigens, especially weakly
expressed antigens.
[0006] Immunological checkpoint proteins are key targets in tumor
therapy and play a very important role in immunotherapy, thus may
be a powerful weapon for conquering cancer. Therefore the accuracy
of detection is required for diagnosis and treatment of cancer.
[0007] In the process of diagnosis and determination of the
following treatment regimen (especially targeted therapy) for
clinical cancer patients, flow cytometry has an incomparable
advantage in determination of subdivision and cell surface markers,
because it can simultaneously detect multiple markers on a single
cell. However, the tumor tissue needs to be treated with a
digestive enzyme into a single cell suspension before it can be
used for subsequent flow cytometry. In the present disclosure, we
have found that the current commercial human tumor digestive agents
or digestive agents commonly used such as collagenases all have a
significant impact on the expression level of various proteins
concerned in the present disclosure (including immunological
checkpoint proteins such as Tim-3 and Lag3), which undoubtedly
increased the risk of misdiagnosis of the disease and easily lead
to wrong treatment. Therefore, the digestive effect of dissociation
reagent on tumor tissues and the protective effect thereof on cell
surface antigen remain to be improved. It is important to find a
digestive enzyme or mixture that is effective in digesting human
tumor tissues and does not affect the expression of surface
markers.
SUMMARY
[0008] In one aspect, the present disclosure relates to a tumor
dissociation reagent.
[0009] Wherein, the tumor dissociation reagent which does not
comprise collagenase but comprises hyaluronidase, said tumor
dissociation reagent does not degrade or partially degrade membrane
surface receptor.
[0010] The present disclosure also relates to a tumor dissociation
reagent which further does not comprise trypsin and collagenase and
comprises hyaluronidase, wherein the tumor dissociation reagent
does not degrade or partially degrade membrane surface
receptor.
[0011] Wherein, the concentration range of the hyaluronidase
described in an embodiment of the present disclosure is preferably
1 mg/mL or less, and the concentration of hyaluronidase is more
preferably 1 .mu.g/mL to 1 mg/mL.
[0012] The present disclosure further relates to the preceding
tumor dissociation reagent, further comprising DNase I.
[0013] Wherein, the concentration range of the DNase I described in
the present disclosure is preferably 50 .mu.g/mL or less, and the
concentration of DNase I is more preferably 1 .mu.g/mL to 50
.mu.g/mL.
[0014] In one embodiment, the tumor dissociation reagent does not
degrade or partially degrade membrane surface receptor, wherein
said membrane surface receptor is at least one checkpoint receptor
selected from the group consisting of receptor CD8, PD-1, PD-L1,
TIM-3 and LAG-3 protein.
[0015] In one embodiment, the proceeding membrane surface receptor
is a checkpoint receptor.
[0016] In a further aspect, the present disclosure provides use of
said tumor dissociation reagent in detecting protein expression
level of an immunological checkpoint marker in tumor tissue.
[0017] In one embodiment of the present disclosure, the preceding
tumor dissociation reagent further comprises DNase I.
[0018] In one embodiment of the present disclosure, the tumor
tissue includes tumor infiltrating immune cell.
[0019] In an embodiment of the present disclosure, the protein
expression in tumor tissue is detected by flow cytometry.
[0020] In an embodiment of the present disclosure, the protein is
membrane surface receptor for checkpoint.
[0021] In an embodiment of the present disclosure, in flow
cytometry of tumor tissue, said membrane surface receptor is at
least one checkpoint receptor selected from the group consisting of
CD8, PD-1, PD-L1, TIM-3 and LAG-3 protein.
[0022] In a further aspect, the present disclosure relates a kit
for tumor dissociation comprising the tumor dissociation reagent of
the present disclosure.
[0023] In an embodiment of the present disclosure, the preceding
tumor dissociation reagent further comprises DNase I.
[0024] The present disclosure also relates to use of the kit in
detecting protein expression in tumor tissue by flow cytometry.
[0025] In a further aspect, the present disclosure also provides a
preparation method, wherein the method may prevent degradation of
immunological checkpoint markers in tumor tissue, wherein cells are
treated with the tumor dissociation agent disclosed by the present
disclosure.
[0026] In one embodiment of the present disclosure, a method of
tumor dissociation for flow cytometry comprises dissolving a tumor
tissue with the tumor dissociation reagent of the present
disclosure.
[0027] In one embodiment of the present disclosure, the tumor
tissue further includes tumor infiltrating immune cell.
Advantageous Effects of the Invention
[0028] The benefit of the present disclosure resides in the
establishment of a tumor dissociation reagent which does not
comprise collagenase or trypsin and does not degrade or partially
degrade membrane surface marker in tumor tissue, therefore the
tumor dissociation reagent is useful in flow cytometry for
detecting the expression level of proteins in tumor tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a flow chart showing the checkpoint proteins PD-1,
Tim-3, and Lag-3 in helper T cells (CD4+) treated with different
digestive enzymes.
[0030] FIG. 2 is a flow chart showing the checkpoint proteins PD-1,
Tim-3, and Lag-3 in cytoxic T cells (CD8+) treated with different
digestive enzymes.
[0031] FIG. 3 is a graph showing the percentages of cell surface
checkpoint proteins after different digestive enzymes
treatment.
[0032] FIG. 4 is a graph showing the percentages of cell surface
checkpoint proteins PD-1, Tim-3 and Lag-3 after treatment with
two-enzyme mixture.
[0033] FIG. 5 is a flow chart showing the cell surface checkpoint
proteins CD4 and CD8 after treatment with different digestive
enzymes.
[0034] FIG. 6 is a graph showing the cell yield after digestion
with different enzymes.
DETAILED DESCRIPTION
[0035] The present disclosure is further described by the specific
embodiments and experimental results. Although specific terms are
used hereinafter for the purpose of clarity, these terms are not to
be limiting the scope of the present disclosure.
[0036] As used herein, the term "dissociation reagent" refers to an
enzymatic digestion reagent, and in the present disclosure, a tumor
dissociation reagent refers to an enzymatic digestion reagent that
digests tumor tissue into a single cell suspension with an enzyme
digestion solution.
[0037] As used herein, the term "membrane surface receptor" refers
to one molecule or a class of molecules on cell surface that may
recognize, bind to a specific biologically active substance
(referred to as a ligand), and the resulting complex may activate
and initiate a series of physical and chemical changes that lead to
the final biological effects of the substance. Changes in the
various factors of the cell environment result in corresponding
changes of the physiological processes within the cell through the
role of the cell membrane receptors.
[0038] The experimental methods in the following examples, unless
otherwise specified, are conventional methods.
EXAMPLES
Example 1
Preparation of Digestive Enzyme Reagents
[0039] Dissociation buffer system of Hyaluronidase: 50 .mu.L of a
solution of hyaluronidase with an initial concentration of 10 mg/mL
(Hyaluronidase, available from Sigma, Cat. No. H3506) was added to
4.95 mL of DMEM medium (the final concentration of hyaluronidase is
100 .mu.g/mL) and then formulated into 5 mL of dissociation reagent
for tumor tissues.
[0040] Dissociation buffer system of Collagenase D: 500 .mu.L of a
solution of collagenase D (Collagenase D, available from Roche
Corporation, Cat. No. 11088882001) with an initial concentration of
10 mg/mL was added to 4.5 mL of DMEM medium (the final
concentration of collagenase D is 1 mg/mL) and then formulated into
5 mL of dissociation reagent for tumor tissues.
[0041] Dissociation buffer system of DNase I: 50 .mu.L of a
solution of DNase I (DNase I, available from Sigma Corporation,
Cat. No. DN25-1G) with an initial concentration of 5 mg/mL was
added to 4.95 mL of DMEM medium (the final concentration of DNase I
is 0.05 mg/mL) and then formulated into 5 mL of dissociation
reagent for tumor tissues.
[0042] Dissociation buffer system of three-enzyme mixture: 50 .mu.L
of a solution of hyaluronidase at the initial concentration of 10
mg/mL, 500 .mu.L of a solution of collagenase D with an initial
concentration of 10 mg/mL and 50 .mu.L of a solution of DNase I
with an initial concentration of 5 mg/mL were added to 4.4 mL of
DMEM medium and then formulated into 5 mL of dissociation reagent
for tumor tissues.
[0043] Dissociation buffer system of two-enzyme mixture: 50 .mu.L
of a solution of hyaluronidase with an initial concentration of 10
mg/mL and 50 .mu.L of a solution of DNase I with an initial
concentration of 5 mg/mL were added to 4.9 mL of DMEM medium and
then formulated into 5 mL of dissociation reagent for tumor
tissues.
[0044] Dissociation buffer system of Miltenyi Human Tumor
Dissociation Kit (Human tumor dissociation kit, Cat. No.
130-095-929): according to the instructions, the storage solution
of digestive enzymes A, H, and R with appropriate concentrations
are formulated, and then stored at -20.degree. C. In the
experiment, 200 .mu.L of storage solution of enzyme H, 100 .mu.L of
storage solution of enzyme R and 25 .mu.L of storage solution of
enzyme A were added to 4.7 mL of DMEM medium and then formulated
into 5 mL of dissociation solution for tumor tissues.
Example 2
Design of Flow Staining
[0045] Specific designs of different staining channels on cell
surface are shown in Table 1.
TABLE-US-00001 TABLE 1 Channels used for cell surface molecular
markers Channel Blank Isotype 2 Panel 2 FITC -- Isotype Tim3 PE --
Isotype PD-1 PerCP -- CD4 CD4 PE-Cy7 -- Isotype Lag-3 APC -- CD3
CD3 APC-Cy7 -- CD8 CD8 BV421 Live/Dead Live/Dead Live/Dead BV510 --
CD45 CD45
Example 3
Effects of Different Digestive Enzymes on the Positive Rate of
Checkpoint Proteins PD-1, TIM3 and LAG-3
[0046] The expression of PD-1, Tim-3 and Lag-3 in cytoxic T cells
(CD8+T) and helper T cells (CD4+T) was induced by PHA treatment.
Whether or not different digestive enzymes will affect expression
levels of the three proteins was analyzed in these two groups of
cells.
[0047] The tumor is not a single cell suspension, thus it cannot be
used in flow cytometry directly. If mechanical dissociation is used
rather than enzymatic dissociation, single-cell yield is relatively
low, therefore positive rate of single cell molecular marker
proteins obtained by mechanical dissociation may not be able to
represent the real value of the whole tissue. Thus, in the present
disclosure, peripheral blood mononuclear cells were used in the
detection of molecular marker proteins of cells. It is confirmed
that some dissociation reagent has an effect on the expression
level of marker proteins on cell surface.
[0048] First, cryopreserved human peripheral blood mononuclear
cells (PBMC) were revived and then treated with 10 .mu.g/mL of PHA
for 48 hours to allow the cells to be activated, followed by
counting. The cells were aliquoted into 21 tubes; the number of
cells is 3.times.10.sup.5 cells per tube. 5 mL of dissociation
buffer was added into each tube, while 5 mL of DMEM medium
(available from Gibco, Cat. No. 11960-051) was added into negative
control tube. The tubes were put into a 37.degree. C. water bath
(available from Shanghai Yiyou Company, model THZ-82), and the
cells were digested for 15 minutes. The specific information of
different treatment groups are as followed:
TABLE-US-00002 TABLE 2 Dissociation conditions in different
treatment groups Name Dissociation Reagent Condition PBMC Negative
control 37.degree. C., 15 minutes Miltenyi Human Tumor Dissociation
Kit 37.degree. C., 15 minutes (Kit) Three-enzyme mixture 37.degree.
C., 15 minutes 1 mg/mL Collagenase D 37.degree. C., 15 minutes 100
.mu.g/mL Hyaluronidase 37.degree. C., 15 minutes 0.05 mg/mL DNase I
37.degree. C., 15 minutes Two-enzyme mixture 37.degree. C., 15
minutes
[0049] The dissociated cells were centrifuged with a centrifuge
(available from Eppendorf, model 5810R) and the supernatant was
removed. The pellets were washed twice with a phosphate buffer PBS
(available from Hyclone Corporation, Cat. No. SH3002802B) and
centrifuged to remove the supernatant, and then incubated with
formulated antibody mixture at 4.degree. C. for 30 minutes in
dark.
[0050] The cells were centrifuged at 4.degree. C., 300.times.g to
remove the supernatant. The cells were resuspended in 200 .mu.L of
staining buffer for flow cytometry (available from BD Co., Cat. No.
Pharmingen-554657) and centrifuged at 4.degree. C., 300.times.g for
5 minutes, and repeated once.
[0051] The cells were re-suspended in 100 .mu.L of cell fixation
buffer (available from BD, Cat. No. BD-554655) and incubated at
4.degree. C. for 20-30 minutes in dark.
[0052] The cells were re-suspended in 200 .mu.L of staining
solution (available from BD Co., Cat. No. Pharmingen-554657),
centrifuged at 4.degree. C., 300.times.g for 5 minutes and repeated
once, and finally re-suspended in staining buffer for flow
cytometry (available from BD Company, Cat. No. Pharmingen-554657),
and the suspension was transferred to a flow tube with a final
volume of 500 .mu.L and detected with a cytometer (BD FACS Canto
II).
[0053] The expression levels of Tim-3 and Lag-3 proteins in T cells
(CD4+) and cytoxic T cells (CD8+) were significantly decreased
after treatment with Miltenyi Human Tumor Dissociation Kit for 15
minutes as compared with the negative control. Similarly, the
expression levels of Lag-3 protein in helper T cells (CD4+) and
cytoxic T cells (CD8+) were significantly decreased after treatment
with three-enzyme mixtures or collagenase (as shown in FIG. 1, FIG.
2, FIG. 3 and TABLE 3).
[0054] In contrast, the expression of different cell surface
molecular markers such as PD-1, Tim-3, and Lag-3 in the
hyaluronidase or DNase I treatment group was unchanged or not
significantly decreased as compared with the negative control group
(as shown in FIG. 1, FIG. 2, FIG. 3 and TABLE 3). This suggests
that the treatment of collagenase or Miltenyi Human Tumor
Dissociation Kit will affect the expression levels of one or more
molecular markers on cell surface such as Tim-3 and Lag-3, which is
disadvantageous for further detection in flow cytometry. But the
reagents not containing collagenase, such as hyaluronidase or DNase
I alone did not affect the expression of these immune cell surface
molecular markers under given conditions. This experiment
demonstrated that neither DNase I at given dose nor hyaluronidase
affected the expression level of the checkpoint proteins.
Furthermore, we found that the results of 1 mg/mL hyaluronidase or
1 .mu.g/mL hyaluronidase treatment were consistent with that of 100
.mu.g/mL hyaluronidase treatment that neither one affected the
level of cell surface molecular markers such as CD8, PD-1, Tim-3
and Lag-3. Similarly, the expression of PD-1, Tim-3 and Lag-3 were
not reduced after treatment with up to 50 .mu.g/mL of DNase I.
Although the fluorescence signal of CD8 was significantly reduced
and PD-1 positive rate was slightly increased, the fluorescence
signal intensities of other immunological checkpoints were not
decreased. And treatment with less than 50 .mu.g/mL of DNase I did
not affect the detection of molecular markers.
[0055] To further verify the protective effect of the hyaluronidase
group or DNase I on the cell surface molecular markers, a mixture
of DNase I and hyaluronidase was used to treat human peripheral
blood mononuclear cells (PBMC) according to above experimental
method. The experimental results indicated that the mixture of
DNase I and hyaluronidase did not affect the expression level of
the checkpoint proteins (as shown in FIG. 4 and Table 4). This data
was also advantageous in the increased selectivity for enzyme
species. It is anticipated that in certain tumor samples, the cell
yield can be increased while the protein expression level is
altered.
TABLE-US-00003 TABLE 3 Positive expression rates (%) of different
cell surface molecular markers PD- PD- Tim- Tim- Lag- Lag- Group
CD4+ CD8+ 1+/CD4+ 1+/CD8+ 3+/CD4+ 3+/CD8+ 3+/CD4+ 3+/CD8+ Negative
55.80 22.80 53.30 58.40 30.80 38.20 29.90 65.00 control Kit 49.10
27.10 59.90 62.60 7.99 11.70 2.74 13.50 Three- 53.70 25.80 58.30
60.50 31.40 35.40 6.05 22.10 enzyme mixture Collage- 53.50 25.20
59.30 60.60 29.70 31.40 5.98 19.80 nase D Hyaluron- 56.10 23.60
45.70 51.80 30.20 37.80 28.70 55.90 idase DNase I 54.10 25.90 48.90
53.10 31.00 34.80 27.40 57.10
TABLE-US-00004 TABLE 4 Positive expression rate (%) of different
cell surface molecular markers after treatment with the two-enzyme
mixture PD- PD- Tim- Tim- Lag- Lag- Group CD4+ CD8+ 1+/CD4+ 1+/CD8+
3+/CD4+ 3+/CD8+ 3+/CD4+ 3+/CD8+ Negative 33.7 25.9 59 49.4 40.6
55.2 53.2 79.9 control Two- 42.2 24 68.7 52.2 39.9 64.5 46.1 76.5
enzyme mixture
Example 4
Detection of Surface Marker CD8 on Human Peripheral Blood
Mononuclear Cells (PBMC)
[0056] Cryopreserved human peripheral blood mononuclear cells
(PBMC) were revived and then treated with 10 .mu.g/mL of PHA for 48
hours to allow the cells to be activated, followed by counting. The
cells were aliquoted, the number of cells is 3.times.10.sup.5 cells
per tube. 5 mL of dissociation buffer was added into each tube,
while 5 mL of DMEM medium (available from Gibco, Cat. No.
11960-051) was added into negative control tube. The tubes were put
into a 37.degree. C. water bath (available from Shanghai Yiyou
Company, model THZ-82), and the cells were digested for 60
minutes.
[0057] The other steps were the same as that in Example 3, and it
was found that the fluorescence intensity of CD8 was decreased
after the treatment with the commercial human tumor kit (Accumax
Cell Dissociation Solution) for 60 minutes, indicating that the kit
treatment reduced the expression level of CD8 (as shown in FIG. 5
and Table 5).
TABLE-US-00005 TABLE 5 Positive expression rates (%) of cell
surface molecular markers CD4 and CD8 after treatment with Kit
Group CD4+ CD8+ Negative control 55.80 22.80 Kit; 60 minutes 53.70
13.90
Example 5
Treatment of Human Tumor Tissues
[0058] Clinical samples obtained by operation were placed in
prepared MACS tissue preservation solution and transported to WuXi
AppTec Co. Ltd. (Shanghai). at 4.degree. C. These tumor samples
were treated within 48 hours after surgery. Before treatment of
clinical tumor tissues, numbering of the corresponding sample was
carried out, and the medical history of the patient, texture and
color of the tumor tissues, and clinical information were recorded.
Tumor tissues were weighed.
[0059] First, the obvious adipose tissue, fibrous tissue and
necrotic part were removed, and then the resulting clinical tumor
samples were washed three times in pre-cooled DMEM medium, finally
the tumor tissue were cut into 10 mm.sup.3 small pieces with
ophthalmic scissors and tweezers.
[0060] 50 .mu.L of a solution of hyaluronidase with an initial
concentration of 10 mg/mL was added to 4.95 mL of DMEM medium (the
final concentration is 100 .mu.s/mL) to prepare the dissociation
reagent for tumor tissue, and then 5 mL of prepared dissociation
reagent for tumor tissue was put into a C tube dedicated for
gentleMACS Dissociator (available from Miltenyi Company, Cat. No.
130-093-237), and the cut tumor tissues were also transferred to
the C tube dedicated for Miltenyi tissue treatment with tweezers.
After tightening of the lid, the tissue fragments in the
dissociation reagent were gently shaken. Tumor tissues within the
range of 10 mg to 1000 mg can be treated with this dissociation
system.
[0061] The C tube was gently inserted into the C-tube slot of the
gentleMACS Dissociator (available from Miltenyi Company, Cat. No.
130-093-235). And it should be noted that the tumor tissue
fragments should be concentrated at the blade area within the C
tube.
[0062] The program was set to the h_tumor_01, and then run once.
The C tube was removed after the end of the program h_tumor_01 and
placed upward for a while so as to place all tumor fragments in the
dissociation solution at the bottom of the tube. If necessary, the
lid can be removed and the tissue adhered on the lid can be
transferred with tweezers to the bottom dissociation reagent. The
removed C tube was put into a 37.degree. C. constant temperature
water bath for 7 minutes, which can be shook appropriately for
several times during the period. Repeat the above steps once.
[0063] The above C tube was gently inserted into the C-tube slot of
the Miltenyi Tissue processor. The program was set to h_tumor_02,
and then run twice. The C tube was removed and the tissue
dissociation was re-suspended in 20 mL of phosphate buffer. A 70
.mu.m cell strainer (available from falcon, Cat. No. 352350) was
placed on a 50 mL centrifuge tube and the dissociated tissue was
re-suspended and slowly passed through the 70 .mu.m cell strainer
and, if necessary, the minced fine tissue pieces can be grounded on
the strainer to obtain more single-cell suspensions. The cell
strainer was washed with 20 mL to 30 mL of phosphate buffer so that
the final volume of the single-cell suspension obtained through the
strainers was 50 mL.
[0064] The cells were centrifuged at 300.times.g for 10 minutes,
and the supernatant was removed with a pipette.
[0065] The single cells obtained in the previous step were
re-suspended in 40 mL of phosphate buffer and were centrifuged at
300.times.g for 7 min.
[0066] The cells were re-suspended into a single-cell suspension
with 0.5 to 5 mL of flow cytometry staining buffer, and counted by
staining of trypan blue.
Example 6
Comparison of Cell Digestion Rates in Different Enzyme-Treated
Groups
[0067] The clinically obtained patient tumor samples were
dissociated with different digestive enzymes under the same
temperature and time conditions according to the method of Example
5, and the single-cell yield was counted with trypan blue staining.
Each sample was counted three times, and the average number+SEM of
single cells per gram of tumor was shown in the figure.
[0068] In FIG. 6, although the cell yield varies depending on the
tumor species or states between different samples, it is comparable
between the combination of the three enzymes involved in the
present disclosure and the conventional enzyme combination methods
reported in the literature, and the differences of cell yield were
not large, thus the resulting cells were sufficient for further
flow cytometry analysis.
[0069] Although the present disclosure is not limited thereto, it
will be understood by those skilled in the art that various
modifications and variations can be made within the scope of the
present disclosure, the manner of changes are also within the scope
of the present disclosure.
* * * * *