CN109666626A

CN109666626A - Single celled method is separated from sample

Info

Publication number: CN109666626A
Application number: CN201811595504.7A
Authority: CN
Inventors: 吴文铭; 彭俊雅; 陈澔; 黄丹; 刘路路; 洪夏飞; 丛林; 李冬晶; 赵玉沛
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2019-04-23

Abstract

The present invention relates to unicellular separation field, discloses and separate single celled method from sample.It is digested the described method includes: normal tissue sample is mixed with digestive juice, the digestive juice contains VIII Collagenase Type, neutral proteinase, trypsin inhibitor and deoxyribonuclease, wherein, the ratio between the VIII Collagenase Type, neutral proteinase, trypsin inhibitor and the enzyme activity unit of deoxyribonuclease is 100-312.5:1-5:5000-25000:1.The present invention can realize that single celled high yield and high motility rate are extracted in the case where total digestion time is shorter, especially pancreatic tissue, capture the unicellular yield of existing pancreatic tissue and the lower problem of motility rate.

Description

Single celled method is separated from sample

Technical field

The present invention relates to unicellular separation fields, and in particular to single celled method is separated from sample.

Background technique

Pancreas is the organ that endocrine and exocrine function are had both in human body.It is thin comprising islet cells, acinus in histology Multiple cellular components such as born of the same parents, vessel cell and interstitial cell.Pancreas generates in the pancreatic juice of secretion containing trypsase, pancreas fat A variety of digestive ferments such as enzyme, amylopsin.With the rise of single cell analysis technology in recent years, people couple and human tissue organ Research is mixed from heterogeneity, and the global tissue analysis interfered with each other is gradually deep into the individual cells for constituting histoorgan Research, importance are self-evident.And in pancreas research field, due to pancreas organ's generally softness in fragile, histology Complicated component and it is easy to happen autodigestion because of potent pancreatin, so that pancreatic single cell research often stops at first Step --- namely the single celled acquisition of pancreatic tissue for analysis.

Human body or the unicellular separation method of animal model tissue organ are drawn from initial capillary, again to airflow classification To micro-fluidic technologies, numerous single-cell techniques more or less all achieves more satisfied knot in different tissues Fruit, but it is then barely satisfactory in pancreatic tissue.By consulting literatures it is found that researcher is slender in separating mouse cancer of pancreas at present When born of the same parents, the enzymic digestion dissociating method based on II Collagenase Type is mostly used greatly, and still, pancreatic single cell researcher indicates in pancreas Greatly challenge is encountered when the unicellular separation of gland or unicellular yield is not high or Cell viability is lower, therefore, exploitation The preparation or method for the pancreatic single cell that can be efficiently separated have particularly important meaning.

Summary of the invention

The purpose of the invention is to overcome the problems, such as that unicellular yield and Cell viability of the existing technology are lower, mention For separating single celled method in a kind of new slave sample.

To achieve the goals above, the present invention provides one kind separates single celled method from sample, this method comprises: Normal tissue sample is mixed with digestive juice and is digested, the digestive juice contains VIII Collagenase Type, neutral proteinase, pancreas egg White enzyme inhibitor and deoxyribonuclease, wherein the VIII Collagenase Type, neutral proteinase, trypsin inhibitor and Ratio between the enzyme activity unit of deoxyribonuclease is 100-312.5:1-5:5000-25000:1.

Through the above technical solutions, the present invention can realize single celled high yield in the case where total digestion time is shorter It is extracted with high motility rate, is based particularly on the unicellular separation of pancreatic tissue, has captured the unicellular yield of existing pancreatic tissue and work The lower problem of rate specifies direction for the single celled separation of pancreatic tissue, may advantageously facilitate to the further of pancreatic tissue Research.

Detailed description of the invention

Fig. 1 is to separate single celled result figure from pancreatic tissue using the method for specific embodiment according to the present invention；

Fig. 2 to Fig. 5 is to separate single celled result figure from pancreatic tissue using different reference methods respectively.

Specific embodiment

The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more New numberical range, these numberical ranges should be considered as specific open herein.

In the present invention, in the absence of explanation to the contrary, term " enzyme activity unit " reaction used is that enzyme contains Amount number, refer under given conditions, converted in 1 minute in 1 micromole substrate or substrate needed for the related group of 1 micromole Enzyme amount, referred to as an enzyme activity unit (IU, also known as U).

Wherein, the definition of the enzyme activity unit of VIII Collagenase Type is: under conditions of pH 7.5 and 37 DEG C, hydrolysis in 5h It is an enzyme activity unit that collagen, which generates and is equivalent to the enzyme amount of the L-Leu of 1 μm of ol,.

The definition of the enzyme activity unit of neutral proteinase is: under conditions of pH 7.5 and 37 DEG C, hydrolyzing junket egg per minute The white enzyme amount for generating the Folin positive amino acid or peptide that are equivalent to 1 μm of ol tyrosine is an enzyme activity unit.

The definition of the enzyme activity unit of trypsin inhibitor is: can inhibit the vigor of a trypsase enzyme activity unit A referred to as inhibitor enzyme activity unit (U), under conditions of pH 7.5 and 25 DEG C, each second hydrolyzes the N- benzoyl-of 1 μm of ol L-arginine ethyl ester (BAEE) is a trypsase enzyme activity unit.

The definition of the enzyme activity unit of deoxyribonuclease is: under conditions of pH 8.0 and 37 DEG C, energy in 10 minutes Enzyme amount needed for the pBR322 Plasmid DNA of enough degradable 1 μ g is an enzyme activity unit.

It is provided by the invention that single celled method is separated from sample includes: to be mixed into normal tissue sample and digestive juice Row digestion, the digestive juice contain VIII Collagenase Type, neutral proteinase, trypsin inhibitor and deoxyribonuclease.

In the method, the ratio between VIII Collagenase Type and the enzyme activity unit of deoxyribonuclease is 100- 312.5:1, as 100:1,120:1,130:1,150:1,180:1,200:1,220:1,240:1,260:1,280:1,300:1, Arbitrary value between 310:1,312:1,312.5:1 or above-mentioned numerical value.

In the method, the ratio between neutral proteinase and the enzyme activity unit of deoxyribonuclease is 1-5:1, such as Arbitrary value between 1:1,1.5:1,2:1,2.5:1,3:1,3.5:1,4:1,4.5:1,5:1 or above-mentioned numerical value.

In the method, the ratio between trypsin inhibitor and the enzyme activity unit of deoxyribonuclease is 5000-25000:1, as 5000:1,8000:1,9000:1,10000:1,12000:1,15000:1,18000:1,20000:1, Arbitrary value between 22000:1,24000:1,25000:1 or above-mentioned numerical value.

Clostridiopetidase A is that unique one kind can degrade the protease of the natural collagen fibre with three strands of superhelixes, this Collagenous fibres are widely present in connective tissue.Clostridiopetidase A mainly by the fermented culture of clostridium histolyticum, is extracted, is refined And obtain, it can also extract and obtain from pig pancreas.In the present invention, the clostridiopetidase A is VIII Collagenase Type, for example, can be The Cat:C2139 of Sigma.

In the present invention, the specific type of neutral proteinase and deoxyribonuclease is not required particularly, Ke Yiwei The common various selections in this field.

Preferably, the neutral proteinase is Dispase II.For example, the Cat:4942078001 of Roche.

Preferably, the deoxyribonuclease is DNaseI.For example, the Cat:M0303S of NEB.

In the present invention, trypsin inhibitor can inhibit trypsase and chymotrypsin, prevent the active egg of other in pancreas The activation of white proenzyme and itself activation of trypsinogen, can choose trypsin inhibitor commonly used in the art.Example Such as, the Cat:T6522 of Sigma.

In the present invention, the digestive juice contains solvent.Relative to every milligram of VIII Collagenase Type, the content of the solvent Can be 0.1-12.5mL, preferably 0.2-3mL, as 0.2mL, 0.4mL, 0.5mL, 0.8mL, 1mL, 1.2mL, 1.3mL, Arbitrary value between 1.5mL, 2mL, 2.5mL or above-mentioned numerical value.

In the present invention, the solvent can be various common preservations or the reagent of dissolution enzyme preparation, it is preferable that described molten Agent is the phosphate buffer containing 4-10 volume % fetal calf serum.

In the present invention, the dosage of the digestive juice is not required particularly, as long as the sample can be submerged.It is excellent It is 1cm relative to volume in the case of choosing³Normal tissue sample, the dosage of the digestive juice is 10-60mL, as 10mL, 15mL, 20mL, 25mL, 26mL, 27mL, 28mL, 30mL, 32mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL or above-mentioned number Arbitrary value between value.

In the present invention, in order to preferably be digested, the volume of the normal tissue sample is preferably 1-2cm × 1-2cm × 0.5-2cm, such as 1.5cm × 1.5cm × 0.5cm.

In the present invention, the digestion can carry out under normal conditions.Preferably, the condition of the digestion includes that temperature is It is 35-38 DEG C, such as any between 35 DEG C, 36 DEG C, 36.5 DEG C, 36.8 DEG C, 37 DEG C, 37.2 DEG C, 37.5 DEG C, 38 DEG C or above-mentioned numerical value Value.

Preferably, the condition of the digestion further include the time be 5-80min, as 5min, 10min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 65min, 70min, 75min, 80min or above-mentioned numerical value it Between arbitrary value.

In the present invention, it is adhered pockets of treatments of the sample in order to further speed up, it can be with shear sample and/or by suction pipe (such as Wu Junbushi suction pipe) blows and beats sample.It filters after a period of time can also be digested through cell sieve, is mended into indigested sample Continue to digest after adding digestive juice.That is, the mode of the digestion can be with are as follows: mix sample with first part digestive juice, shear Sample, and the sample after shearing is mixed with second part digestive juice, sample is blown and beaten, cell sieve is crossed；Indigested sample is added Digestive juice continues digestion repeatedly, crosses cell sieve collection respectively and adds the digestion product obtained after digestive juice every time.Relative to volume For 1cm³Normal tissue sample, the dosage of first part's digestive juice can be 400-1000 μ L.It is 1cm relative to volume³Just Normal tissue sample, the dosage of second part digestive juice can be 5-20mL.It is 1cm relative to volume³Normal tissue sample, often The dosage of the secondary digestive juice added can be 5-20mL.

In the present invention, in order to further promote the progress of digestion, the method also includes: before digestion, in tissue preserration Fiber, fat and the necrotic tissue of normal tissue sample are removed in liquid.

Wherein, the dosage of the tissue preserration liquid is not required particularly, as long as the sample can be submerged.Institute Stating tissue preserration liquid can be conventional selection, for example, can be the serum-free cell containing fetal calf serum and protease inhibitors Freezing media.

In the present invention, the method also includes: postdigestive sample is contacted with erythrocyte cracked liquid to crack red thin Born of the same parents obtain the unicellular of separation after washing.Postdigestive sample be cell suspending liquid, be centrifuged and take precipitating, precipitate with it is red Cell pyrolysis liquid is contacted to remove red blood cell.The dosage of the erythrocyte cracked liquid can be conventional selection, for example, relative to body Product is 1cm³Normal tissue sample, the dosage of the erythrocyte cracked liquid can be 3-8ml.Washing lotion used in washing can also Conventional selection is thought, for example, can be the phosphate buffer containing 0.02-0.08 weight % bovine serum albumin.It is washed to described The dosage of liquid is not particularly limited, for example, being 1cm relative to volume³Normal tissue sample, the dosage of the washing lotion can be with For 3-8ml.

A kind of specific embodiment according to the present invention, the described method comprises the following steps:

1) in tissue preserration liquid, the fiber cut on normal tissue sample, fat and downright bad group are pruned with Sterile ophthalmic It knits；

2) sterile PBS washing；

3) sample is put into sterile centrifugation tube, it (is 1cm relative to volume that first part's digestive juice, which is added,³Normal group Tissue samples, the dosage of first part's digestive juice can be 400-1000 μ L)；

4) it is cut using Sterile ophthalmic and tissue is cut into clast (about 2 × 2 × 1mm³), it operates on ice, shear time is no more than 5 Minute；

5) tissue debris is transferred in sterile petri dish, it (is 1cm relative to volume that second part digestive juice, which is added,³'s Normal tissue sample, the dosage of second part digestive juice can be 5-20mL)；

6) it is blown and beaten using Wu Junbushi suction pipe, collects supernatant and cross cell sieve, take filtrate, indigested tissue debris, which is added, to disappear Change liquid continue to digest in sterile petri dish, until tissue digestion completely or obtain satisfied normal tissue it is unicellular (relative to Volume is 1cm³Normal tissue sample, the dosage for the digestive juice added every time can be 5-20mL)；

7) filtrate obtained in step 6) is centrifuged；

8) abandon supernatant, be added lysate softly blow and beat, be resuspended precipitating, be protected from light at room temperature split it is red, if there is cotton-shaped indissoluble object, Cell is crossed again weed out remove；

9) re-suspension liquid is transferred in sterile centrifugation tube and is centrifuged；

10) supernatant is abandoned, first part's washing lotion is added and softly blows and beats resuspension precipitating, it is meticulous again if there is cotton-shaped indissoluble object Born of the same parents, which weed out, to be removed, and re-suspension liquid is transferred to new sterile centrifugation tube and continuously adds second part washing lotion (first part's washing lotion and the The volume ratio of two part washing lotions can be 1:4-5)；

11) it is centrifuged；

12) step 10) is repeated；

13) it is centrifuged.

In the present invention, the method can also include using cell culture medium to digesting resulting unicellular cultivate. The cell culture medium can be the conventional medium for expanding cell, or the dimensional culture base containing growth factor, So that the single cells grown obtained is three-dimensional organoid model.

In the present invention, the normal tissue sample can be common tissue sample, can be pancreatic samples, such as tumour The normal pancreatic tissue at place position.It is unicellular method of the invention is particularly suitable for being separated from pancreatic samples, therefore, according to The preferred embodiment of the present invention, the normal tissue sample are the normal pancreatic tissue at position where tumour.

The present invention will be described in detail by way of examples below.

Table 1

Table 2

Embodiment 1

The present embodiment is used to illustrate of the invention single from normal pancreatic tissue (Normal Pancreas at position where tumour) separation The method of cell, wherein used tissue preserration liquid, digestive juice, lysate and washing lotion are as shown in table 1, in every liter of digestive juice The enzyme activity unit value and concentration of each ingredient are as shown in table 2, and the preparation method of digestive juice are as follows: mixing VIII Collagenase Type, Dispase II and trypsin inhibitor dissolve mixing powder using solvent, add DNaseI mixing.

Sample is divided into five groups and is tested and (be shown in Table 3), specific steps are as follows:

1) in tissue preserration liquid, it is (normal that fiber, fat and necrotic tissue cut on sample etc. is pruned with Sterile ophthalmic Pancreatic tissue block size about 1.5 × 1.5 × 0.5cm³, color is yellow, and quality is medium partially soft, and surface is cut without black burnt necrosis, too many blood The visible clearly pancreas leaflet in face separates, and derives from pancreatic neoplasm patient, and operation cuts acquisition, patient's signed informed consent Book).

2) sterile PBS is washed 3 times.

3) sample is put into 5ml sterile EP tube (eppendorf), about 500 μ l of digestive juice is added.

4) it is cut using Sterile ophthalmic and tissue is cut into clast (about 2 × 2 × 1mm³), it operates on ice, shear time is no more than 5 Minute.

5) tissue debris is transferred in 10cm sterile petri dish, 10ml digestive juice is added, be placed in 37 DEG C of incubator digestion.

6) it is mixed once every 5min using the piping and druming of Wu Junbushi suction pipe, and is seen under 10 × 20,10 × 40 power microscopes Examine cell dissociation situation.There is individual cells suspension in visible digestive juice under the microscope when first 10min, by digestive juice It is collected into sterile 50ml centrifuge tube together with tissue debris, is stood after soft piping and druming, collect supernatant and cross 40 μm of cell sieve (cell Strainer), filtrate is taken.Indigested tissue debris adds digestive juice 10ml and continues to digest in 10cm sterile petri dish, directly It completely or obtains that satisfied pancreas normal tissue is unicellular (altogether continue 50min) to tissue digestion, first 10min is collected Filtrate is given it up, and because it contains more impurity, separates unicellular difficulty.The filtrate that later every 10min digests can be distinguished It retains, carries out aftermentioned operating procedure respectively, finally choose motility rate, the optimal single cell suspension sample of cell quantity.

7) filtrate 800g obtained in step 6) is centrifuged 4min (using horizontal rotor).

8) supernatant is abandoned, 5ml lysate is added and softly blows and beats, precipitating is resuspended, is protected from light splits red 5min (if there is cotton-shaped difficulty at room temperature Molten object then crosses 40 μm of cell sieve removals again).

9) re-suspension liquid is transferred in sterile 15ml centrifuge tube and is centrifuged, centrifugal force: 800g, time: 4min.

10) supernatant is abandoned, addition 1ml washing lotion softly blows and beats resuspension precipitating and (if there is cotton-shaped indissoluble object, crosses 40 μm of cells again Weed out and remove), re-suspension liquid is transferred to a new sterile 15ml centrifuge tube and continuously adds 5ml washing lotion.

11) 600g is centrifuged 5min.

12) step 10) is repeated.

13) 400g is centrifuged 6min.

14) supernatant is abandoned, precipitating is resuspended in appropriate washing lotion, and (precipitating of covering 15ml centrifugation bottom of the tube, uses 100 μ l washing lotion weights It is outstanding), take isometric cell and trypan blue to mix, microscopy cellular morphology, motility rate, impurity situation etc., cell counter (life Cell countess) detection Cell viability (living cells quantity accounts for the percentage of total number of cells in=every ml product) is (every with concentration Total number of cells in ml product, including dead cell), as a result as shown in Fig. 1-5 and table 3.

In Fig. 1-5, upper figure is the result figure for observing digestion effect after digesting under ordinary optical microscope (40 ×), the following figure For the result figure for observing Cell viability after further diluting under ordinary optical microscope (40 ×).

Table 3

From Fig. 1 and table 3 as can be seen that can effectively be separated from normal tissue using method of the invention it is unicellular, Basic soilless sticking, Cell viability is 85% or more.

From Fig. 2, Fig. 3 and table 3 as can be seen that if the ratio in digestive juice between each component is not in the scope of the present invention Interior, cell, which exists, after digestion reunites, and Cell viability is relatively low.

From Fig. 4 and table 3 as can be seen that can be obtained using VIII Collagenase Type more significantly more than other types of clostridiopetidase A Unicellular and Cell viability it is also higher.

From Fig. 5 and table 3 as can be seen that being only used cooperatively VIII Collagenase Type, neutral proteinase, trypsin inhibitor Single cells population (reuniting few) and Cell viability can be effectively improved with deoxyribonuclease, four kinds of ingredients are indispensable, Collaboration plays a role.

Embodiment 2

The present embodiment is used to illustrate of the invention from the single celled method of pancreas source tumor tissues separation, wherein is made Tissue preserration liquid, digestive juice, lysate and washing lotion are as shown in table 1, each in every liter of digestive juice (preparation method is with embodiment 1) The enzyme activity unit value and concentration of a ingredient are as shown in the I-1 of table 2.

1) it in tissue preserration liquid, is pruned with Sterile ophthalmic and cuts pancreatic neoplasm sample (1.5 × 1.5 × 0.5cm³, operation Cut acquisition, patient's signed informed consent form) on fiber, fat and necrotic tissue etc..

2) sterile PBS is washed 3 times.

5) tissue debris is transferred in 50ml sterile tube, 15ml digestive juice is added, be placed in 37 DEG C, 50rmp/min shakes It is digested on bed.

6) pockets of tissue debris piping and druming will be adhered using Wu Junbushi suction pipe after 5min to mix.

7) continue to observe tissue clastic state after digesting 5min, if being still adhered agglomerating, repeatedly step 6) -7), if tissue Clast dispersion then continues to digest 20min, and total digestion time is no more than 30min.

8) digestion finishes, and stands 3min after reusing Wu Junbushi suction pipe piping and druming tissue debris, collects supernatant and cross 40 μm Cell sieve (cell strainer), obtains filtrate and is placed on ice.

9) indigested tissue adds 15ml digestive juice, continues after digesting 20min, blows and beats tissue using Wu Junbushi suction pipe Clast crosses 40 μm of cell sieves together with digestive juice and tissue debris, obtains filtrate.

10) merge the filtrate obtained twice, 800g is centrifuged 4min (using horizontal rotor).

11) supernatant is abandoned, 5ml lysate is added and softly blows and beats, precipitating is resuspended, is protected from light splits red 5min (if having cotton-shaped at room temperature Indissoluble object then crosses 40 μm of cell sieve removals again).

12) re-suspension liquid is transferred in sterile 15ml centrifuge tube and is centrifuged, centrifugal force: 800g, time: 4min.

13) supernatant is abandoned, addition 1ml washing lotion softly blows and beats resuspension precipitating and (if there is cotton-shaped indissoluble object, crosses 40 μm of cells again Weed out and remove), re-suspension liquid is transferred to a new sterile 15ml centrifuge tube and continuously adds 5ml washing lotion.

14) 600g is centrifuged 5min.

15) step 13) is repeated.

16) 400g is centrifuged 6min.

17) supernatant is abandoned, precipitating is resuspended in appropriate washing lotion, and (precipitating of covering 15ml centrifugation bottom of the tube, uses 100 μ l washing lotion weights It is outstanding), take isometric cell and trypan blue to mix, microscopy cellular morphology, motility rate, impurity situation etc., cell counter (life Cell countess) Cell viability and concentration are detected, it is as a result similar to Example 1, it can be effectively using method of the invention Separated from tumor tissues unicellular, basic soilless sticking, Cell viability is 80% or more；And if ratio between each component Not within the scope of the invention, after digestion there is reunion in cell, and Cell viability is relatively low；And it can be obtained using VIII Collagenase Type It is also higher to obtain the unicellular and Cell viability more significantly more than other types of clostridiopetidase A.In addition, in the feelings for lacking a kind of ingredient Under condition, single cells population and Cell viability are undesirable, further illustrate that four kinds of ingredients are indispensable, collaboration plays a role.

The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to Protection scope of the present invention.

Claims

1. one kind separates single celled method from sample, which is characterized in that this method comprises: by normal tissue sample and digestion Liquid mixing is digested, and the digestive juice contains VIII Collagenase Type, neutral proteinase, trypsin inhibitor and deoxyribose Nuclease, wherein the enzyme activity of the VIII Collagenase Type, neutral proteinase, trypsin inhibitor and deoxyribonuclease Ratio between unit of force is 100-312.5:1-5:5000-25000:1.

2. according to the method described in claim 1, wherein, the neutral proteinase is Dispase II；

And/or the deoxyribonuclease is DNaseI.

3. method according to claim 1 or 2, wherein relative to every milligram of VIII Collagenase Type, in the digestive juice The content of solvent is 0.1-12.5mL.

4. according to the method described in claim 3, wherein, the solvent is slow for the phosphate containing 4-10 volume % fetal calf serum Fliud flushing.

5. according to the method described in claim 1, being 1cm relative to volume wherein³Normal tissue sample, the digestive juice Dosage is 10-60mL.

6. method according to claim 1 or 5, wherein the volume of the normal tissue sample be 1-2cm × 1-2cm × 0.5-2cm。

7. according to the method described in claim 1, wherein, the condition of the digestion includes: that temperature is 35-38 DEG C, time 5- 80min。

8. according to the method described in claim 1, wherein, the method also includes: before digestion, removed in tissue preserration liquid Fiber, fat and the necrotic tissue of normal tissue sample.

9. according to the method described in claim 8, wherein, the tissue preserration liquid is to contain fetal calf serum and protease inhibitors Serum-free cell freezing media.

10. according to claim 1, method described in 8 or 9, wherein the method also includes: by postdigestive sample and red thin The contact of cellular lysate liquid obtains the unicellular of separation to splitting erythrocyte after washing.