CN110468030A - A kind of animal tissue digestion collection device and method - Google Patents
A kind of animal tissue digestion collection device and method Download PDFInfo
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- CN110468030A CN110468030A CN201910831553.4A CN201910831553A CN110468030A CN 110468030 A CN110468030 A CN 110468030A CN 201910831553 A CN201910831553 A CN 201910831553A CN 110468030 A CN110468030 A CN 110468030A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/22—Settling tanks; Sedimentation by gravity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
Abstract
The present invention relates to a kind of animal tissues to digest collection device, including agitating paddle, digestion cup, mesh basket and receiving flask, the mesh basket is located in digestion cup, electric heating metallic sheath is provided on the lateral wall of the digestion cup, the lower part of the digestion cup is connected with connecting tube, the side of the connecting tube is connected with drainage tube, the drainage tube is provided with the first valve on the part of connecting tube, the connecting tube, which is located on the part below drainage tube, is provided with the second valve, the side of the lower end connection collecting pipe of the connecting tube, one end of the collecting pipe is extend into receiving flask；It is provided with third valve in the first collection pipe portion, is provided with peristaltic pump in the second collection pipe portion；The receiving flask, collecting pipe at least first collection pipe portion, at least the second connecting section of connecting tube be located in ice bath bucket.Do mechanization operation may be implemented in the present invention, to reduce the influence of manual work, reduces operational pollution, improves cell changes and yield.
Description
Technical field
The present invention relates to animal tissue's digestion and isolated technical fields, and in particular to a kind of animal tissue's digestion collection dress
It sets and method.
Background technique
Cell transplantation is an important directions of transplantation surgeries technology development, or will faced there are many cell products
It is applied on bed.By taking pancreatic islets transplantation as an example, diabetes are to seriously threaten the disease of human health, and pancreatic islets transplantation can pass through
It adjusts endogenous insulin secretion and treats diabetes.Since source of people donor is insufficient, it is unable to satisfy large quantities of transplanting demands, pork insulin
With actrapid monotard's only poor amino acid, using having longer history with diabetic, and pig can carry out gene
Modification reduces immunological rejection after transplanting, therefore current pig pancreas islet has entered clinic as the transplanting source of heterograft
Experimental stage.
The method separated and collected in the prior art to porcine islet is as follows: firstly, taking the neonatal pig in 2~5 days
After Patients Under Ketamine Anesthesia, sterile taking-up pancreas is used containing moving back after the washing of 100U mycillin cleaning solution to super-clean bench, removes pancreas
Gland perienchyma；Then, it is packed into centrifuge tube after shredding, injects 1mg/1ml Collagenase V digestive juice, centrifuge tube is placed in constant temperature
It is rocked in water-bath to sand shaped, the time takes around 7-10min；Subsequently, it moves into 4 DEG C of addition in super-clean bench and contains 20%
The hanks liquid of FBS stops digestion；Subsequently, using 1-500 μm of strainer filtering, the islet tissue agglomerate of filtration is collected.For
The Collagenase V for not digesting tissue and bulk connective tissue, adding in right amount with concentration for not passing through sieve carries out 2 digestion, directly
Pass through sieve to substantially all of tissue.Reuse the HBSS tissue washout liquid centrifuge washing tissue containing 10%FBS three times, then
Carry out the training of pancreas islet.This method is relied on substantially and is manually completed, from the point of view of the experimental result of this team early period, from disappearing
Change time 2.14 ± 0.31h/ of the average out to item to culture, although 3-5 pancreas can be handled simultaneously, the time of digestion can prolong
It is long, cause overall processing time to extend；It is 2.12 ± 0.53 ten thousand/item that islet tissue, which harvests quantity, and equivalent is 1.91 ± 0.35
Ten thousand IEQ/ items, average activity are 91.23 ± 3.21%, and are easy to appear pollution, excessive the problems such as digesting.Simultaneously because artificial
The otherness of operation also can make collection process the difference of Different Results occur.Needed in the standard of present Islets Xenotransplantation compared with
The pig pancreas islet of multi-quantity, thus the method efficiency is lower.
Summary of the invention
The invention is intended to provide a kind of animal tissue digestion collection device and method, digested by mechanical device, point
From and collect, to reduce manual operation, reduce operational pollution, improve cell changes and yield.The invention solves skill
Art problem is achieved through the following technical solutions.
A kind of animal tissue's digestion collection device, including agitating paddle, digestion cup, mesh basket and receiving flask, improvements exist
In: the mesh basket is located in digestion cup, and upper end opening, side wall and the bottom of the mesh basket are sieve, the digestion cup top
With openable and closable cup lid, the agitating paddle passes through cup lid and extend into mesh basket, is provided with electricity on the lateral wall of the digestion cup
The lower part of thermometal set, the digestion cup is connected with connecting tube, and the side of the connecting tube is connected with drainage tube, the drainage tube
It is provided with the first valve on the part of connecting tube, the connecting tube, which is located on the part below drainage tube, is provided with the second valve
Door, the side of the lower end connection collecting pipe of the connecting tube, one end of the collecting pipe is extend into receiving flask；The connecting tube
Part between digestion cup and the second valve is the first connecting section, the part between the second valve and collecting pipe is
Second connecting section, the collecting pipe are in that part between receiving flask and connecting tube is the first collection pipe portion, remainder is
Second collects pipe portion；It is provided with third valve in the first collection pipe portion, is provided with peristaltic pump in the second collection pipe portion；
The receiving flask, collecting pipe at least first collection pipe portion, at least the second connecting section of connecting tube be located in ice bath bucket.
Preferably, the aperture of the sieve is 100-1000 μm.
Preferably, the mesh basket is placed in digestion cup by hook, and mesh basket is caught in one end of hook, the other end is affiliated to and is disappearing
At the rim of a cup for changing cup.
Preferably, the lower part of the digestion cup is funnel-shaped and is connected in funnelform exit with connecting tube.
The method that animal tissue's digestion is collected is carried out using above-mentioned apparatus the present invention also provides a kind of.
A kind of animal tissue's digestion collection method, thes improvement is that: using as preceding any described device progress is as follows
Step:
S1, ice water is added in ice bath bucket, ice water forms the first liquid level in ice bath bucket, and the first liquid level at least submerges collection
Bottle；
S2, the first valve is in close state, the second valve is in the open state, third valve be in close shape
State opens peristaltic pump, the external collection liquid of collecting pipe is imported into collecting pipe, in connecting tube using peristaltic pump, so that collection liquid
The second liquid level is formed in connecting tube, second liquid level is between the second valve and drainage tube；
S3, digestive juice is added from the rim of a cup of digestion cup, so that digestive juice forms third liquid level in digestion cup, described the
Three liquid levels at least submerge part mesh basket, are layered using the density of collection liquid greater than the density and discontinuous density gradient of digestive juice
Principle, so that digestive juice and collection liquid form layering at the second liquid level；
S4, electric heating metallic sheath is opened, starts to heat the digestive juice in digestion cup；
S5, animal tissue to be digested is shredded and is put into mesh basket；
S6, agitating paddle is opened, animal tissue to be digested is digested while stirring, made by the animal tissue of sieve in gravity
Collecting pipe is settled down under to be in the part between third valve and peristaltic pump, and with collection continue gradually build up to connection
In second connecting section of pipe；
S7, according to the accumulation of animal tissue in collecting pipe and connecting tube, close the second valve, open third valve and compacted
Dynamic pump, peristaltic pump by collecting pipe collection liquid and animal tissue be delivered in receiving flask, complete the digestion of an animal tissue
It collects.
Preferential, it further include step S8, S9,
S8, third valve and peristaltic pump are closed, opens the second valve；
S9, step S5, S6, S7, S8 are repeated, until completing the continuous digestion collection work of animal tissue.
It preferably, further include step S10, S11, S12,
S10, after the completion of continuously digestion is collected, the second valve is closed, peristaltic pump is inverted the second company of collecting pipe, connecting tube
Collection liquid in adapter tube portion is imported into the external the first container of collecting pipe, peristaltic pump is closed, by collecting pipe from the first container
It takes out and is connected with third container；
S11, the first valve is opened, digests the digestive juice in cup, connecting tube is located at the digestion in the part on drainage tube top
Liquid enters in the external second container of drainage tube along drainage tube；
S12, it opens the second valve, open peristaltic pump, the digestive juice and collection liquid in connecting tube enter in collecting pipe simultaneously
The peristaltic pump being inverted is imported into the external third container of collecting pipe, closes peristaltic pump, to complete in digestion collection device
The recycling of liquid.
Preferably, the first liquid level in the ice bath bucket at least submerges the second connecting section of connecting tube.
Preferably, in S4, the temperature of heating is controlled at 30-42 DEG C.
Preferably, in S5, animal tissue to be digested shreds into 1-5mm3Fritter；
Preferably, in S6, the revolving speed of agitating paddle is 30-1000rpm.
One of present invention animal tissue digests collection device, and the stirring of agitating paddle can accelerate animal tissue in mesh basket
Be digested speed, sieve will not digest completely and the biggish animal tissue of volume intercepts in continuing to digest in mesh basket, disappears
Histocyte group after change is through sieve and with row sedimentation under the action of gravity, successively by the upper layer digestive juice in connecting tube
With the collection liquid of lower layer, enter in collecting pipe；Peristaltic pump can rotate forward collection liquid is imported into collecting pipe, will be in collecting pipe
Collection liquid is imported into receiving flask, can also be inverted and be exported to the liquid in collecting pipe in external container；Drainage tube and
The setting of one valve can drain into the digestive juice digested in cup and connecting tube in external container, realize the recycling of digestive juice
It utilizes.
It is provided using the ice water of ice bath bucket and its interior splendid attire for receiving flask and collects the temperature that histocyte or cell mass are suitable for.
The height of second liquid level is in the second valve and drainage tube, and digestive juice is flowed out from drainage tube after being easy to use, and is carried out
Recycling；It, can will be in the second connecting section and collecting pipe of connecting tube using the reversion of peristaltic pump meanwhile after closing the second valve
Collection liquid is recycled.Digestive juice in digestion cup at least submerges part mesh basket, can be disappeared while stirring to animal tissue
Change, sieve will not digest completely and the biggish animal tissue of volume intercept in continue digestion in mesh basket or the later period carry out it is clear
It removes.The temperature of a constant temperature is provided using electric heating metallic sheath for the digestive juice in digestion cup.It, can using the forward and backward of peristaltic pump
To import collection liquid into collecting pipe, the liquid in collecting pipe can also be exported.
Compared with prior art, do mechanization operation may be implemented in the present invention, to reduce the influence of manual work, reduces dirty
It contaminates, improve efficiency；Meanwhile being collected using the digestion of mechanization, the quality collected to animal tissue's digestion can be improved.
Detailed description of the invention
Fig. 1 is the structural diagram of the present invention one；
Fig. 2 is structural schematic diagram two of the invention；
Appended drawing reference in attached drawing is successively are as follows: 1, agitating paddle, 2, cup lid, and 3, digestion cup, 4, mesh basket, 5, electric heating metallic sheath,
6, connecting tube, the 7, first valve, 8, drainage tube, the 9, second valve, 10, third valve, 11, peristaltic pump, 12, collecting pipe, 13, receipts
Collection bottle, 14, ice bath bucket, the 15, first liquid level, the 16, second liquid level, 17, third liquid level, 18, the first container, 19, second container,
20, third container.
Specific embodiment
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
Embodiment 1:
A kind of animal tissue's digestion collection device, including agitating paddle 1, digestion cup 3, mesh basket 4 and receiving flask 13, improve it
It is in and is located in digestion cup 3 in: the mesh basket 4, upper end opening, side wall and the bottom of the mesh basket 4 are sieve, the digestion
3 tops of cup have openable and closable cup lid 2, and the agitating paddle 1 passes through cup lid 2 and extend into mesh basket 4, the outside of the digestion cup 3
Electric heating metallic sheath 5 is provided on wall, the lower part of the digestion cup 3 is connected with connecting tube 6, and the side of the connecting tube 6 is connected with
Drainage tube 8, the drainage tube 8 are provided with the first valve 7 on the part of connecting tube 6, and the connecting tube 6 is located at drainage tube 8
The second valve 9, the side of the lower end connection collecting pipe 12 of the connecting tube 6, the collecting pipe 12 are provided on the part of lower section
One end extend into receiving flask 13；The part that the connecting tube 6 is between digestion cup 3 and the second valve 9 is the first connection
Pipe portion, the part between the second valve 9 and collecting pipe 12 are the second connecting section, and the collecting pipe 12 is in receiving flask 13
Part between connecting tube 6 is the first collection pipe portion, remainder is the second collection pipe portion；It is set in the first collection pipe portion
It is equipped with third valve 10, is provided with peristaltic pump 11 in the second collection pipe portion；At least the of the receiving flask 13, collecting pipe 12
One collection pipe portion, at least the second connecting section of connecting tube 6 are located in ice bath bucket 14.
The stirring of agitating paddle 1 can accelerate the speed that is digested of animal tissue in mesh basket 4, and sieve will not digest completely and body
The biggish animal tissue of product intercepts in continuing digestion in mesh basket 4 or the later period is purged, and postdigestive histocyte group is saturating
It crosses sieve and is settled with going under the action of gravity, successively pass through the collection liquid of the upper layer digestive juice and lower layer in connecting tube 6, into
Enter into collecting pipe 12；Peristaltic pump 11, which can be rotated forward, to be imported into collection liquid in collecting pipe 12, leads the collection liquid in collecting pipe 12
Enter into receiving flask 13, can also invert and export to the liquid in collecting pipe 12 in external container；Drainage tube 8 and the first valve
The setting of door 7 can drain into the digestive juice digested in cup 3 and connecting tube 6 in external container, realize the recycling of digestive juice
It utilizes.
Embodiment 2:
On the basis of embodiment 1, the aperture of the sieve is 100-1000 μm.
The aperture of sieve is set as 100-1000 μm, can filter the indigested tissue of removal and connective tissue.
Embodiment 3:
On the basis of embodiment 1 or 2, the mesh basket 4 is placed in digestion cup 3 by hook, and net is caught in one end of hook
Basket 4, the other end are affiliated at the rim of a cup of digestion cup 3.
This gives mesh baskets 4 to be set to a kind of fixed form in digestion cup 3, can by linking up with fixed mesh basket 4
To facilitate the disassembly of mesh basket 4 conveniently to be cleaned, sterilized to mesh basket 4 and digestion cup 3 in turn.
Embodiment 4:
On the basis of embodiment 1-3 is any, it is described digestion cup 3 lower part it is funnel-shaped and in funnelform exit with
Connecting tube 6 is connected.
It is funnel-shaped to digest 3 lower part of cup, the tissue in digestive juice is drained using funnelform inclined surface, is conveniently disappeared
Histocyte or cell mass after change enter in connecting tube 6 along funnel-form inner wall.
Embodiment 5:
The side that animal tissue's digestion is collected is carried out using such as any described device of embodiment 1-4 this gives a kind of
Method.
A kind of animal tissue digests collection method, thes improvement is that: using as any described device of embodiment 1-4 into
Row following steps:
S1, ice water is added in ice bath bucket 14, ice water forms the first liquid level 15 in ice bath bucket 14, and the first liquid level 15 at least soaks
Do not have receiving flask 13；
S2, the first valve 7 is in close state, the second valve is 9 in the open state, third valve 10 is in and closes
State opens peristaltic pump 11, and the external collection liquid of collecting pipe 12 is imported into collecting pipe 12, in connecting tube 6 using peristaltic pump 11,
So that collection liquid forms the second liquid level 16 in connecting tube 6, second liquid level 16 is between the second valve 9 and drainage tube 8；
S3, digestive juice is added from the rim of a cup of digestion cup 3, so that digestive juice forms third liquid level 17, institute in digestion cup 3
It states third liquid level 17 and at least submerges part mesh basket 4, density and discontinuous density level bands using the density of collection liquid greater than digestive juice
The principle for spending layering, so that digestive juice and collection liquid form layering at the second liquid level 16；
S4, electric heating metallic sheath 5 is opened, starts to heat the digestive juice in digestion cup 3；
S5, animal tissue to be digested is shredded and is put into mesh basket 4；
S6, agitating paddle 1 is opened, animal tissue to be digested is digested while stirring, by the animal tissue of sieve in gravity
It is settled down to collecting pipe 12 under effect to be in the part between third valve 10 and peristaltic pump 11, and continues gradually to accumulate with collection
Tire out into the second connecting section of connecting tube 6；
S7, according to the accumulation of animal tissue in collecting pipe 12 and connecting tube 6, close the second valve 9, open third valve
10 and peristaltic pump 11, peristaltic pump 11 by collecting pipe 12 collection liquid and animal tissue be delivered in receiving flask 13, complete primary
The digestion of animal tissue is collected.
It is that receiving flask 13 provides collection histocyte or cell mass is suitable for using the ice water of ice bath bucket 14 and its interior splendid attire
Temperature.The height of second liquid level 16 is in the second valve 9 and drainage tube 8, and digestive juice is from drainage tube 8 after being easy to use
Outflow, is recycled；It, can be by the second connecting tube of connecting tube 6 using the reversion of peristaltic pump 11 meanwhile after closing the second valve 9
Collection liquid in portion and collecting pipe 12 is recycled.Digestive juice in digestion cup 3 at least submerges part mesh basket 4, can be to animal
Tissue is digested while stirring, sieve will not digest completely and the interception of volume biggish animal tissue in continue in mesh basket 4 into
Row digestion or later period are purged.The temperature of a constant temperature is provided using electric heating metallic sheath 5 for the digestive juice in digestion cup 3.Benefit
With the forward and backward of peristaltic pump 11, collection liquid can be imported into collecting pipe 12, the liquid in collecting pipe 12 can also be exported.
In separation for the purpose of cell or tissue etc. is to purify, it is often used sucrose isodensity gradient well prepared in advance
Liquid is separated according to the density contrast of cell or tissue.Its working principle is that: respectively with certain speed between different densities particle
Sedimentation forms the method for zone on different densities gradient region.Density gradient media should be pre-formed, the density gradient of medium
Range will include the density of the tissue particles to be collected.For density gradient if it is discontinuous, group is woven in the close of similar density
Spend interface overlapping；Density gradient is also possible to continuous density gradient, this like cell can according to the accurate density of gradient liquid into
Row efficiently separates.
In the present embodiment, collection liquid can be using such as publication number CN109468266A, entitled animal and human pancreas' organ
The washing solution gone out given in tissue, cell washing solution and preparation method after digestion, can also be using such as publication number
CN105886454A, it is entitled it is a kind of tissue and cell purification density gradient solution formula given in go out density gradient it is molten
Liquid.It is greater than the density of digestive juice using the density of collection liquid, can guarantees that digestive juice and collection liquid are stablized at the second liquid level 16
Layering.The collection solution is added to the common ingredient for increasing solution density, such as Sodium Amidotrizoate, Iodixanol, and is usually used in
Both continuously and discontinuously density gradient separation, density can preferably maintain 1.026-1.060g/ml to the islet tissue of humans and animals,
And preferable separating interface, component density gradient are formed with the lower buffer of remaining density or culture solution.
Embodiment 6:
It further include step S8, S9 on the basis of embodiment 5,
S8, third valve 10 and peristaltic pump 11 are closed, opens the second valve 9；
S9, step S5, S6, S7, S8 are repeated, until completing the continuous digestion collection work of animal tissue.
Increased two steps in the present embodiment can be realized the continuous digestion and collection operation of animal tissue, improve
The collection efficiency of animal tissue.
Embodiment 7:
It further include step S10, S11, S12 on the basis of embodiment 6,
After the completion of S10, continuous digestion are collected, close the second valve 9, the reversion of peristaltic pump 11 is by collecting pipe 12, connecting tube 6
Collection liquid in second connecting section is imported into the external the first container 18 of collecting pipe 12, peristaltic pump 11 is closed, by collecting pipe
12 take out from the first container 18 and connect with third container 20；
S11, the first valve 7 is opened, digests the digestive juice in cup 3, connecting tube 6 is located in the part on 8 top of drainage tube
Digestive juice enters in the external second container 19 of drainage tube 8 along drainage tube 8；
S12, it opens the second valve 9, open peristaltic pump 11, the digestive juice and collection liquid in connecting tube 6 enter collecting pipe
In 12 and the peristaltic pump 11 that is inverted is imported into the external third container 20 of collecting pipe 12, peristaltic pump 11 is closed, to complete
Digest the recycling of liquid in collection device.
The recycling to the liquid in digestion collection device may be implemented in increased three steps in the present embodiment.
Embodiment 8:
On the basis of embodiment 5-7 is any, the first liquid level 15 in the ice bath bucket 14 at least submerges the of connecting tube 6
Two connecting section.
The second connecting section that first liquid level 15 at least submerges connecting tube 6 is set, and the ice water that can use ice bath bucket 14 is
Second connecting section of connecting tube 6, collecting pipe 12 provide the temperature for collecting that histocyte or cell mass are suitable for.
Embodiment 9:
On the basis of embodiment 5-8 is any, in S4, the temperature of heating is controlled at 30-42 DEG C.
Embodiment 10:
On the basis of embodiment 5-9 is any, in S5, animal tissue to be digested shreds into 1-5mm3Fritter.
Embodiment 11:
On the basis of embodiment 5-10 is any, in S6, the revolving speed of agitating paddle 1 is 30-1000rpm.
Embodiment 12:
On the basis of embodiment 5-11 is any, by taking the separation of neonatal pig pancreas islet as an example.
Step S1, S2, S3 are carried out, the heating and temperature control of electric heating metallic sheath 5 is at 38-42 DEG C in S4；In S5, take 2~5 days
After Patients Under Ketamine Anesthesia, sterile taking-up pancreas is used to be moved back containing the washing of 100U mycillin cleaning solution to ultra-clean interior neonatal pig
Platform is put into the digestion cup 3 for being loaded with the Collagenase V containing 1mg/1ml after shredding and closes digestion cup 3 with cup lid 2；In S6, stirring
The revolving speed of paddle 1 is 1000rpm；Postdigestive pancreatic cell group penetrates 100 μm in digestion cup 3 of sieve as the effect of gravity is heavy
It is down to the interface of digestive juice and collection liquid, is i.e. at the second liquid level 16, then goes through the collection liquid that density is 1.026g/ml
In, and accumulated in the second connecting section of collecting pipe 12 and connecting tube 6.Due to collection liquid density be greater than digestive juice density and
The principle of discontinuous density gradient layering, digestive juice are layered in the top of collection liquid, and pancreatic cell/cell mass density, which is greater than, to be received
The density of liquid collecting, so can smoothly accumulate through collection liquid and in the second connecting section of collecting pipe 12 and connecting tube 6, pancreas
Gland cell/cell mass is collected liquid removal pancreatic cell/cell mass surface during through the process and accumulation of collection liquid
Digestive juice, have the function that stop digestion.Step S7 is carried out according to pancreatic cell/cell mass accumulation, it is thin to complete pancreas
The collection of born of the same parents/cell mass.The neonatal pig pancreas islet of collection directly carries out tissue cultures after centrifuge washing twice.
Using the present apparatus and method, the time of operation is substantially reduced.Neonatal pig islet tissue from digestion to culture when
Between average out to 1.54 ± 0.28h/ item.Because using the lesser sieve in aperture, obtained islet tissue volume is equal for this separation
Smaller, it is 3.31 ± 0.78 ten thousand/item that islet tissue, which harvests quantity, and equivalent is 2.31 ± 0.47 ten thousand IEQ/ items, and average activity is
92.43 ± 3.55%, and there is not pollution problem.Isolated pancreas islet quantity greatly increases, while activity is also
It improves, organizes no bacteria pollution.
Embodiment 13:
On the basis of embodiment 5-11 is any, by taking the separation of neonatal pig pancreas islet as an example.
Step S1, S2, S3 are carried out, the heating and temperature control of electric heating metallic sheath 5 is at 38-42 DEG C in S4；In S5, take 3 2~
In 5 days new life after pig Patients Under Ketamine Anesthesia, sterile taking-ups pancreas use containing 100U mycillin cleaning solution wash move back to
Super-clean bench is put into the digestion cup 3 for being loaded with the Collagenase V containing 1mg/1ml after shredding and closes digestion cup 3 with cup lid 2；In S6,
The revolving speed of agitating paddle 1 is 30rpm；Postdigestive pancreatic cell, which is rolled into a ball, penetrates the sieve for digesting 1000 μm in cup 3 with the work of gravity
With the interface for being settled down to digestive juice and collection liquid, i.e. at the second liquid level 16, then go through the collection that density is 1.035g/ml
In liquid, and accumulated in the second connecting section of collecting pipe 12 and connecting tube 6.It is carried out according to pancreatic cell/cell mass accumulation
The neonatal pig pancreas islet of step S7, collection directly carry out tissue cultures after centrifuge washing twice.
Using the present apparatus and method, the time of operation is substantially reduced.Time of 3 neonatal pig pancreas from digestion to culture
1.65 ± 0.52h/3 pancreas of average out to, it is 1.98 ± 0.45 ten thousand/pancreas, equivalent 2.55 that islet tissue, which harvests quantity,
The problems such as ± 0.35 ten thousand IEQ/ pancreas, average activity are 93.35 ± 2.53%, tissue no bacteria pollution, excessive digestion.Separation
Obtained pancreas islet equivalent greatly increases, while activity also increases.
Embodiment 14:
On the basis of embodiment 5-11 is any, by taking the separation of neonatal pig pancreas islet as an example.
Step S1, S2, S3 are carried out, the heating and temperature control of electric heating metallic sheath 5 is at 38-42 DEG C in S4；In S5, take 2~5 days
After Patients Under Ketamine Anesthesia, sterile taking-up pancreas is used to be moved back containing the washing of 100U mycillin cleaning solution to ultra-clean interior neonatal pig
Platform is put into the digestion cup 3 for being loaded with the Collagenase V containing 1mg/1ml after shredding and closes digestion cup 3 with cup lid 2；In S6, stirring
The revolving speed of paddle 1 is 500rpm；Postdigestive pancreatic cell group penetrates 500 μm in digestion cup 3 of sieve as the effect of gravity is heavy
It is down to the interface of digestive juice and collection liquid, is i.e. at the second liquid level 16, then goes through the collection liquid that density is 1.060g/ml
In, and accumulated in the second connecting section of collecting pipe 12 and connecting tube 6.It is walked according to pancreatic cell/cell mass accumulation
Rapid S7 completes the collection of pancreatic cell/cell mass.The neonatal pig pancreas islet of collection directly carries out tissue after centrifuge washing twice
Culture.
Using the present apparatus and method, the time of operation is substantially reduced.From digestion to culture time average out to 1.34 ±
0.21h/ item, it is 2.41 ± 0.36 ten thousand/item that islet tissue, which harvests quantity, and equivalent is 2.24 ± 0.52 ten thousand IEQ/ items, average living
Property be 93.12 ± 2.43%, tissue no bacteria pollution, excessively digestion the problems such as.Isolated pancreas islet equivalent, activity also have compared with
It is big to improve.
It is noted that above-mentioned detailed description is all exemplary, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein all has with the application person of an ordinary skill in the technical field's
It is generally understood the identical meaning of institute.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to illustrative embodiments described herein.As used herein, unless the context clearly indicates otherwise, otherwise singular
Form is also intended to include plural form.Additionally, it should be understood that when in the present specification use term "comprising" and/or
When " comprising ", existing characteristics, step, operation, device, component and/or their combination are indicated.
It should be noted that the description and claims of this application and term " first " in above-mentioned attached drawing, "
Two " etc. be to be used to distinguish similar objects, without being used to describe a particular order or precedence order.It should be understood that using in this way
Term be interchangeable under appropriate circumstances, so that presently filed embodiment described herein can be in addition to illustrating herein
Or the sequence other than those of description is implemented.
In addition, term " includes " and " having " and their any deformation, it is intended that cover and non-exclusive include.Example
Such as, the process, method, system, product or equipment for containing a series of steps or units those of are not necessarily limited to be clearly listed
Step or unit, but may include being not clearly listed or intrinsic for these process, methods, product or equipment other
Step or unit.
For ease of description, spatially relative term can be used herein, as " ... on ", " ... top ",
" ... upper surface ", " above " etc., for describing such as a device shown in the figure or feature and other devices or spy
The spatial relation of sign.It should be understood that spatially relative term is intended to comprising the orientation in addition to device described in figure
Except different direction in use or operation.For example, being described as if the device in attached drawing is squeezed " in other devices
It will be positioned as " under other devices or construction after part or construction top " or the device of " on other devices or construction "
Side " or " under other devices or construction ".Thus, exemplary term " ... top " may include " ... top " and
" in ... lower section " two kinds of orientation.The device can also be positioned with other different modes, such as be rotated by 90 ° or be in other orientation, and
And respective explanations are made to the opposite description in space used herein above.
In explanation in detail above, with reference to attached drawing, attached drawing forms a part of this paper.In the accompanying drawings, similar symbol
Number similar component is typically determined, unless context otherwise indicates.In detailed description, attached drawing and claim
Embodiment illustrated described in book is not meant to be restrictive.In the spirit for not departing from theme presented herein
Or under range, other embodiments can be used, and can make other changes.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of animal tissue digests collection device, including agitating paddle (1), digestion cup (3), mesh basket (4) and receiving flask (13),
Be characterized in that: the mesh basket (4) is located in digestion cup (3), and upper end opening, side wall and the bottom of the mesh basket (4) are sieve,
The digestion cup (3) top has openable and closable cup lid (2), and the agitating paddle (1) passes through cup lid (2) and extend into mesh basket (4),
It is provided with electric heating metallic sheath (5) on the lateral wall of digestion cup (3), the lower part of digestion cup (3) is connected with connecting tube
(6), the side of the connecting tube (6) is connected with drainage tube (8), and the drainage tube (8) is arranged on the part of connecting tube (6)
Have the first valve (7), the connecting tube (6), which is located on the part below drainage tube (8), is provided with the second valve (9), the company
The side of lower end connection collecting pipe (12) of (6) is taken over, one end of the collecting pipe (12) is extend into receiving flask (13)；It is described
Connecting tube (6) be in digestion cup (3) and the second valve (9) between part be the first connecting section, be in the second valve (9) and
Part between collecting pipe (12) is the second connecting section, the collecting pipe (12) be in receiving flask (13) and connecting tube (6) it
Between part be the first collection pipe portion, remainder is the second collection pipe portion；Third valve is provided in the first collection pipe portion
Door (10), it is described second collection pipe portion on be provided with peristaltic pump (11)；The receiving flask (13), collecting pipe (12) at least first
Collect pipe portion, at least the second connecting section of connecting tube (6) is located in ice bath bucket (14).
2. a kind of animal tissue according to claim 1 digests collection device, it is characterised in that: the aperture of the sieve is
100-1000μm。
3. a kind of animal tissue according to claim 1 digests collection device, it is characterised in that: the mesh basket (4) passes through
Hook is placed in digestion cup (3), and mesh basket (4) is caught in one end of hook, the other end is affiliated at the rim of a cup of digestion cup (3).
4. a kind of animal tissue according to claim 1 digests collection device, it is characterised in that: digestion cup (3)
Lower part is funnel-shaped and is connected in funnelform exit with connecting tube (6).
5. a kind of animal tissue digests collection method, it is characterised in that: carried out such as using such as any described device of claim 1-4
Lower step:
S1, ice water is added in ice bath bucket (14), ice water forms the first liquid level (15) in ice bath bucket (14), and the first liquid level (15) is extremely
Submerge receiving flask (13) less；
S2, the first valve (7) is in close state, the second valve (9) is in the open state, third valve (10) be in close
Closed state, open peristaltic pump (11), using peristaltic pump (11) by the external collection liquid of collecting pipe (12) imported into collecting pipe (12),
In connecting tube (6), so that collection liquid forms the second liquid level (16) in connecting tube (6), second liquid level (16) is in second
Between valve (9) and drainage tube (8)；
S3, digestive juice is added from the rim of a cup of digestion cup (3), so that digestive juice forms third liquid level (17) in digestion cup (3),
The third liquid level (17) at least submerges part mesh basket (4), is greater than the density of digestive juice and discontinuous using the density of collection liquid
The principle of density gradient layering, so that digestive juice and collection liquid form layering at the second liquid level (16)；
S4, electric heating metallic sheath (5) are opened, starts to heat the digestive juice in digestion cup (3)；
S5, animal tissue to be digested is shredded and is put into mesh basket (4)；
S6, agitating paddle (1) is opened, animal tissue to be digested is digested while stirring, made by the animal tissue of sieve in gravity
Collecting pipe (12) is settled down under to be in the part between third valve (10) and peristaltic pump (11), and with collection continue by
It gradually builds up in the second connecting section of connecting tube (6)；
S7, according to the accumulation of animal tissue in collecting pipe (12) and connecting tube (6), close the second valve (9), open third valve
Door (10) and peristaltic pump (11), peristaltic pump (11) by collecting pipe (12) collection liquid and animal tissue be delivered to receiving flask (13)
In, the digestion for completing an animal tissue is collected.
6. a kind of animal tissue according to claim 5 digests collection method, it is characterised in that: it further include step S8, S9,
S8, third valve (10) and peristaltic pump (11) are closed, opened the second valve (9)；
S9, step S5, S6, S7, S8 are repeated, until completing the continuous digestion collection work of animal tissue.
7. a kind of animal tissue according to claim 6 digests collection method, it is characterised in that: further include step S10,
S11, S12,
After the completion of S10, continuous digestion are collected, close the second valve (9), peristaltic pump (11) is inverted collecting pipe (12), connecting tube
(6) the collection liquid in the second connecting section is imported into the external the first container (18) of collecting pipe (12), closes peristaltic pump
(11), collecting pipe (12) is taken out from the first container (18) and is connected with third container (20)；
S11, the first valve (7) are opened, the digestive juice, connecting tube (6) in digestion cup (3) are located at the part on drainage tube (8) top
Interior digestive juice enters in the external second container (19) of drainage tube (8) along drainage tube (8)；
S12, the second valve (9) are opened, open peristaltic pump (11), the digestive juice and collection liquid in connecting tube (6) enter collection
It manages the peristaltic pump (11) in (12) and being inverted to imported into the external third container (20) of collecting pipe (12), closes peristaltic pump
(11), to complete the recycling of liquid in digestion collection device.
8. a kind of animal tissue according to claim 5 digests collection method, it is characterised in that: in the ice bath bucket (14)
The first liquid level (15) at least submerge the second connecting section of connecting tube (6).
9. a kind of animal tissue according to claim 5 digests collection method, it is characterised in that: in S4, the temperature of heating
Control is at 30-42 DEG C.
10. a kind of animal tissue according to claim 5 digests collection method, it is characterised in that:
Preferably, in S5, animal tissue to be digested shreds into 1-5mm3Fritter；
Preferably, in S6, the revolving speed of agitating paddle (1) is 30-1000rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910831553.4A CN110468030A (en) | 2019-09-04 | 2019-09-04 | A kind of animal tissue digestion collection device and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910831553.4A CN110468030A (en) | 2019-09-04 | 2019-09-04 | A kind of animal tissue digestion collection device and method |
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| CN110468030A true CN110468030A (en) | 2019-11-19 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0512769A2 (en) * | 1991-05-03 | 1992-11-11 | Becton, Dickinson and Company | Device and method for collecting and processing fat tissue and procuring microvessel endothelial cells |
| CN2624925Y (en) * | 2003-06-24 | 2004-07-14 | 中国人民解放军第三军医大学 | Simple extraneous liver perfusion device for digesting and separating liver cells |
| CN102304477A (en) * | 2011-09-02 | 2012-01-04 | 温州医学院 | Large animal pancreatic island digestion and separation device |
| CN203866305U (en) * | 2014-05-09 | 2014-10-08 | 蒋小峰 | Pancreaticislets (langerhans) separating device |
| CN109735447A (en) * | 2019-02-20 | 2019-05-10 | 温州市怡康细胞移植技术开发有限公司 | It is a kind of for separating the devices and methods therefor of islet tissue |
-
2019
- 2019-09-04 CN CN201910831553.4A patent/CN110468030A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0512769A2 (en) * | 1991-05-03 | 1992-11-11 | Becton, Dickinson and Company | Device and method for collecting and processing fat tissue and procuring microvessel endothelial cells |
| CN2624925Y (en) * | 2003-06-24 | 2004-07-14 | 中国人民解放军第三军医大学 | Simple extraneous liver perfusion device for digesting and separating liver cells |
| CN102304477A (en) * | 2011-09-02 | 2012-01-04 | 温州医学院 | Large animal pancreatic island digestion and separation device |
| CN203866305U (en) * | 2014-05-09 | 2014-10-08 | 蒋小峰 | Pancreaticislets (langerhans) separating device |
| CN109735447A (en) * | 2019-02-20 | 2019-05-10 | 温州市怡康细胞移植技术开发有限公司 | It is a kind of for separating the devices and methods therefor of islet tissue |
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