CN109699633A - It is a kind of to cause the freezing protectant of miniflow to import the Cryopreservation of removal micro-system and method and cell based on sound - Google Patents
It is a kind of to cause the freezing protectant of miniflow to import the Cryopreservation of removal micro-system and method and cell based on sound Download PDFInfo
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- CN109699633A CN109699633A CN201910012175.7A CN201910012175A CN109699633A CN 109699633 A CN109699633 A CN 109699633A CN 201910012175 A CN201910012175 A CN 201910012175A CN 109699633 A CN109699633 A CN 109699633A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The freezing protectant of miniflow is caused to import removal micro-system based on sound the invention discloses a kind of, including SAW device, hydrophobic treatment is carried out to the part of the surface of SAW device, hydrophobic surface area is formed, complete freezing protectant importing cell in the hydrophobic surface area and/or removes freezing protectant from cell.Also disclose a kind of freezing protectant importing minimizing technology and a kind of Cryopreservation of cell that miniflow is caused based on sound.The freezing protectant imports the displacement that removal system and method cause miniflow effect to complete freezing protectant and water intracellular using sound, it CPA is imported into intracellular or removes from cell to realize, reduce traditional manual mode of operation osmotic shock and mechanical damage caused by cell.The Cryopreservation can preferably save the form and integrality of cell, improve the survival rate of cell, be suitble to the cryo-conservation of few cells.
Description
Technical field
The invention belongs to cell cryo-conservation technical fields, and in particular to a kind of to cause the freezing protectant of miniflow to lead based on sound
Enter and removes micro-system, a kind of freezing protectant importing minimizing technology importing removal micro-system using the freezing protectant, and
A kind of Cryopreservation for the cell importing minimizing technology using the freezing protectant.
Background technique
In clinical application, a large amount of fresh cells are generally needed to use for medical treatment, this just involves how to cultivate and save
The problem of cell.Cell cryo-conservation (cryopreservation) technology is a kind of method that can effectively extend cell survival,
And form, integrality, the function etc. of cell are not damaged during preservation.One typical case of cell cryo-conservation technology
It is exactly umbilical cord mesenchymal stem cells (umbilical cord matrix mesenchymal stem cells (hUCM-MSCs))
Preservation.Since mescenchymal stem cell has reproducible ability, so that it has a wide range of applications in cell therapy.But
It needs to avoid cell from chromosome abnormality, vicious transformation or function occur impaired such stem cell cryo-conservation before clinical application
Situations such as.
In cryo-conservation technology, need to inhibit cell using freezing protectant (cryoprotective agent, CPA)
The formation of intracellular ice (intracellular ice) in process of freezing and thawing, therefore before carrying out liquid nitrogen cryogenics preservation, it needs
CPA is imported into (loading) into cell.Since CPA has toxicity, before cell enters clinical use, need CPA from thin
(unloading) is completely removed in born of the same parents.Traditional CPA is imported and the method for removal, using test tube, is manually operated to complete.
Specifically, the importing and removal of CPA needs to be divided into multistep realization, and each step is by the CPA solution of various concentration and carefully
Cell lysis liquid is mixed, and is sufficiently mixed solution by manual shaking test tube, inside and outside cell wall the concentration difference of CPA cause CPA to
Intracellular diffusion, replaces water intracellular.By CPA concentration incremented by successively, water intracellular can be thoroughly replaced as CPA.Traditional approach
Manual operation, time-consuming very long, to cell generation osmotic shock and mechanical damage (caused by cell and the collision of imbibition tube wall)
Greatly, so that the form of cell, integrality are destroyed, to reduce the survival rate of cell.It is also possible to introduce manual operation mistake
Accidentally, biological cross contamination etc..Traditional approach uses tube manipulation, therefore is not suitable for operation and the cryoprotection of few cells.
Therefore, system and cell Cryopreservation are imported and removed there is an urgent need to a kind of new efficient CPA to solve
Problem is stated, realizes that the efficient of CPA imports and the fast freezing of removal and cell, is preferably protected during entire cryo-conservation
Form, the integrality for holding cell, improve the survival rate of cell.
Summary of the invention
The freezing protectant of miniflow is caused to import removal micro-system based on sound the object of the present invention is to provide a kind of, the freezing is anti-
It protects agent and imports the displacement that removal micro-system causes miniflow effect to complete freezing protectant and water intracellular using the sound of SAW device,
It realizes and freezing protectant is imported into cell and removes freezing protectant from cell, reduce traditional manual mode of operation to thin
Osmotic shock and mechanical damage caused by born of the same parents.
The freezing protectant of miniflow is caused to import minimizing technology, the freezing based on sound it is a further object of the present invention to provide a kind of
Protective agent imports minimizing technology and above-mentioned freezing protectant importing removal micro-system is utilized, and being divided into multistep will be with the cold of concentration difference
Freeze protective agent to mix with cell culture fluid, reduces traditional manual mode of operation osmotic shock caused by cell and mechanical damage
Wound.
Another object of the present invention is to provide a kind of Cryopreservation of cell, and the Cryopreservation of the cell utilizes
Above-mentioned freezing protectant imports minimizing technology and therefore can preferably save the form and integrality of cell, improve cell
Survival rate.
For achieving the above object, the present invention the following technical schemes are provided:
It is a kind of to cause the freezing protectant of miniflow to import removal system, including SAW device based on sound, to surface acoustic wave
The part of the surface of device carries out hydrophobic treatment, forms hydrophobic surface area, the hydrophobic surface area complete freezing protectant with
The displacement of water intracellular is realized and freezing protectant is imported cell and/or removes freezing protectant from cell.
When the SAW device works, it can surface acoustic wave is generated, on the surface acoustic wave and hydrophobic surface area
When drop meets, surface acoustic wave can be coupled in drop in the form of leaky wave.The energy of surface acoustic wave is decayed rapidly in drop,
The energy of loss is converted into the kinetic energy of liquid, forms liquid stream in liquid internal, this phenomenon is known as sound and causes miniflow.Miniflow is caused in sound
Under the action of, CPA is mixed with cell culture fluid Quick uniform, in the cell under the action of outer freezing protectant concentration difference, freezing
Protective agent is replaced with water intracellular, be can be realized that freezing protectant is imported cell and/or removes the freezing in cell and is protected
Agent.
Specifically, the miniflow actuation capability of the SAW device is realized by Rayleigh miniflow, Rayleigh miniflow
Have the characteristics that leaky wave energy is high, miniflow actuation effect is good, be advantageously implemented CPA and the quick of cell culture fluid is sufficiently mixed,
Quick, the thorough displacement of CPA and water intracellular.Also the wave that can use other can generate sound cause miniflow is realized functionally similar micro-
System, such as Lamb wave and shearing wave (shearing wave can also cause sound to cause miniflow).
Wherein, the piezoelectric layer of the SAW device is single using lithium niobate, lithium tantalate, quartz, zinc oxide, aluminium nitride etc.
Brilliant material or polycrystalline material.The size of SAW device is about 1cm × 1cm, has the characteristics that micromation.
In the present invention, using fluorination treatment or silane (silane) etc. can be deposited on SAW device surface,
To realize the hydrophobic treatment to SAW device part of the surface.The hydrophobic surface of formation is conducive to cell culture fluid on the surface
Smooth movement or generation sound cause miniflow, reduce the mechanical damage of cell, so that the integrality of cell and configuration of surface keep good,
Improve cell survival rate.
Preferably, the SAW resonator includes Piezoelectric Substrates, at least one interdigital transducer, wherein it is described extremely
Few interdigital transducer preparation is in the piezoelectric substrate surface.
Preferably, the driving microwave power of the SAW device is lower than+34dBm.In the driving range of microwave power
It is interior, it drives microwave power adjustable by accuracy controlling and optimization freezing protectant multistep imports and the effect of removal.
Further, the working frequency of the SAW device is 10~100MHz.Lower working frequency facilitates
The interaction of surface acoustic wave and liquid is improved, the attenuation length of surface acoustic wave in a liquid is increased, to enhance miniflow actuating
Performance.Cell wall after freezing is more crisp, and excessively high microwave power will form too fast sound and cause miniflow, to cause the broken of cell wall
Damage.
Further, the surface temperature when SAW resonator works is up to 37 DEG C.The proper temperature is thin
The survival of born of the same parents.In order to reduce the cost of cell freezing and improve the utilization rate of SAW device, the importing and removal of the CPA
The same surface acoustic wave chip can be used.
It is a kind of to cause the freezing protectant of miniflow to import minimizing technology based on sound, comprising the following steps:
(1) above-mentioned freezing protectant is provided and imports removal micro-system, the freezing protectant imports removal micro-system packet
Containing SAW device；
(2) freezing protectant and cell culture fluid containing cell are imported into using dropper or micro-fluidic device described
Hydrophobic surface area on SAW device；
(3) SAW device described in the power drive lower than+34dBm, the SAW device generation sound surface
Wave, under the driving of the surface acoustic wave, the freezing protectant is uniformly mixed with the cell nutrient solution, outer cold in the cell
Freeze under diffusion caused by the concentration difference of protective agent, complete the displacement of freezing protectant and water intracellular, realizes and protect freezing
Agent imports cell or removes freezing protectant from cell.
Specifically, in step (3), it can control SAW resonator described in+27dBm~+34dBm power drive.
The driving power of the range can more accurately freezing protectant multistep import and removal effect.
A kind of Cryopreservation of cell, comprising the following steps:
(1) it imports minimizing technology using above-mentioned freezing protectant freezing protectant is imported into cell；
(2) liquid nitrogen frozen is carried out to the cell for having imported freezing protectant；
(3) water-bath defrosting is carried out to the cell of liquid nitrogen frozen；
(4) minimizing technology is imported using above-mentioned freezing protectant to handle the cell that water-bath is thawed, it will be in cell
Freezing protectant removal.
In the Cryopreservation for the cell that invention provides, the importing of CPA is at least divided into the completion of 2 steps, and illustration can
To be divided into the completion of 3 to 6 steps, the concentration of CPA is incremented by successively.Each step is by the CPA of various concentration and cell culture fluid on sound surface
Mixing is completed in the hydrophobic surface area of wave device, is realized by diffusion with intracellular water using CPA concentration difference inside and outside cell wall
Exchange.As the concentration of CPA is incremented by successively, the final intracellular CPA concentration for obtaining target value.Specifically, step (1) includes:
(1-1) configuration has the freezing protectant of at least two kinds of concentration of concentration difference；
The freezing protectant of minimum concentration and cell culture drop containing cell are passed through dropper or microfluidic devices by (1-2)
Part imported into the hydrophobic surface area of the SAW device, the surface acoustic wave device described in the power drive lower than+34dBm
Part, under the driving of surface acoustic wave, the freezing protectant of minimum concentration is uniformly mixed with cell culture fluid, is caused in 8~12s sound micro-
Under the action of stream, the freezing protectant concentration of intraor extracellular reaches balance, and the freezing protectant of minimum concentration is imported by realization
Into the cell；
(1-3) according to concentration from low to high, using the method for step (1-2), successively by the freezing protectant of various concentration
It imported into intracellular.
In the Cryopreservation for the cell that invention provides, the removal of CPA is also at least divided into the completion of 2 steps, illustrates
Be segmented into 3 to 6 steps completion, CPA removal step number it is more, to cell generate osmotic shock it is smaller, cell integrity and
Survival rate is higher.The CPA of various concentration is completed to mix by each step with cell culture fluid on the surface of SAW device.Cell
The concentration difference of CPA causes CPA to external diffusion inside and outside wall, and realization aqueous solution is exchanged with intracellular CPA's；And with removal step number
Increase, the descending concentrations of CPA completely remove intracellular CPA to zero.Specifically, step (4) includes:
(4-1) configuration has the freezing protectant of at least three kinds of concentration of concentration difference；
The cell culture drop that the freezing protectant of maximum concentration and water-bath thaw is passed through dropper or micro-fluidic device by (4-2)
It is imported into the hydrophobic surface area of the SAW device, the SAW device described in the power drive lower than+34dBm,
Under the driving of surface acoustic wave, the freezing protectant of maximum concentration is uniformly mixed with cell culture fluid, causes miniflow in 8~12s sound
Under the action of, the freezing protectant concentration of intraor extracellular reaches balance, with the freezing protectant in diluting cells；
(4-3) from high to low, using the method for step (4-2), is successively protected using the freezing of various concentration according to concentration
The intracellular freezing protectant of dilution agent, completely removes until by intracellular freezing protectant.
In the Cryopreservation of above-mentioned cell, the importing and removal of CPA can be completed in one minute, quickly high
Effect.
Compared with prior art, the device have the advantages that are as follows:
Freezing protectant provided by the invention imports removal micro-system, causes stream effect using the sound wave of SAW device,
The mixing to freezing protectant and cell can be realized automatically.
Freezing protectant provided by the invention imports minimizing technology, and the importing and removal of CPA is real on SAW device
It is existing, by the driving power of precision control part, the capacity of drop and incorporation time, can automatically, fast and efficiently realize
The displacement of CPA and water intracellular.
The Cryopreservation of cell provided by the invention, above-mentioned freezing protectant importing minimizing technology realization is utilized will
Freezing protectant imports cell and removes the freezing protectant in cell.Compared with traditional manual manipulation mode, the freezing is anti-
Protecting agent importing minimizing technology has osmotic shock small, the low feature of mechanical damage, so that cell integrity and form keep good
Good, cell survival rate greatly improves, can low temperature to avoid human operational error/biology cross contamination, suitable for few cells
Save etc..
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to do simply to introduce, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art, can be with root under the premise of not making the creative labor
Other accompanying drawings are obtained according to these attached drawings.
Fig. 1 be embodiment provide freezing protectant import removal system structural schematic diagram and Rayleigh sound cause it is micro-
Flow schematic illustration；
Fig. 2 is the CPA steps for importing schematic diagram based on single interdigital transducer that embodiment provides；
Fig. 3 is the CPA removal step schematic diagram based on single interdigital transducer that embodiment provides；
Fig. 4 is the CPA steps for importing schematic diagram based on four interdigital transducers that embodiment provides；
Fig. 5 is that the CPA based on four interdigital transducers that embodiment provides removes step schematic diagram；
Fig. 6 is the liquid nitrogen cooling and water-bath defrosting step schematic diagram for the cell that embodiment provides；
Mark explanation: 128 ° of Y-cut LiNbO3Piezoelectric Substrates 1, interdigital transducer 2, hydrophobic surface 3, drop 4, freezing are anti-
Protect agent (CPA) 5, human umbilical cord's matrix mescenchymal stem cell (hUCM-MSCs) 6, microchannel 7, microchannel 8, interdigital transducing
Device 9, interdigital transducer 10, interdigital transducer 11.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, with reference to the accompanying drawings and embodiments to this
Invention is described in further detail.It should be appreciated that the specific embodiments described herein are only used to explain the present invention,
And the scope of protection of the present invention is not limited.
Embodiment 1
Referring to Fig. 1, the freezing protectant importing removal micro-system provided in this embodiment that miniflow is caused based on sound includes sound table
Surface wave device, specifically by 128 ° of Y-cut LiNbO3Monocrystalline piezoelectric substrate 1, the figure being deposited in lithium niobate monocrystal Piezoelectric Substrates 1
Shape interdigital transducer 2 constitutes single-ended SAW resonator.The length of the single-ended SAW resonator is 0.9cm, width
For 0.6cm.
The front intermediate region of interdigital transducer 2 first carries out oxygen plasma treatment, then passes through fluorination treatment on its surface
Or one layer of silane (1H, 1,2h, 2H-perflurodecyltriethoxyl) of evaporation, to complete the processing of hydrophobic surface 3.?
When carrying out the importing and removal of CPA, drop 4 (CPA or cell suspending liquid) is located on hydrophobic surface 3.Hydrophobic surface 3 facilitates liquid
The smooth movement of drop 4 or introducing sound cause miniflow to be rapidly reached the uniform mixing of liquid, reduce the mechanical damage caused by cell.
When carrying out the importing and removal of CPA, the wavelength of removal system design is imported according to freezing protectant, in interdigital transducing
2 both ends of device apply the alternating signal of 10~100MHz, due to inverse piezoelectric effect, 128 ° of Y-cut LiNbO3Monocrystalline piezoelectric substrate 1
Surface can excite Rayleigh wave.When Rayleigh wave and drop 4 meet, Rayleigh wave can be coupled in the form of leaky wave
Into drop 4.The energy of Rayleigh wave is decayed rapidly in drop 4, and the energy of loss is converted into the kinetic energy of liquid, in liquid
Inside forms liquid stream, and this phenomenon is known as sound and causes miniflow.Miniflow effect is caused based on sound, when the mixed liquor of CPA and cell suspending liquid
When for drop 4, just it is able to achieve CPA and is mixed with the Quick uniform of cell suspending liquid.
When 2 both ends of interdigital transducer apply alternating signal, the power of signal is unsuitable excessively high, should be lower than+34dBm.At this
In power bracket, surface temperature and fluid temperature when SAW resonator works are lower than 37 DEG C, conducive to the survival of cell.Letter
When number power is lower, sound causes miniflow speed lower, and liquid mixing is uneven, can cause biggish osmotic shock, cause cell
The breakage of wall.
Embodiment 2
A kind of freezing protectant importing minimizing technology based on single-ended SAW resonator is present embodiments provided, specifically
Removal system is imported using freezing protectant as shown in Figure 1, the single-ended SAW resonator is by 128 ° of Y-cut LiNbO3
Monocrystalline piezoelectric substrate 1, the graphical interdigital transducer 2 being deposited in lithium niobate monocrystal Piezoelectric Substrates 1 are constituted, and pass through fluorination treatment
Form hydrophobic surface 3.
Specifically, it includes freezing protectant being imported into cell, and remove cell that freezing protectant, which imports minimizing technology,
In freezing protectant, detailed process is as follows:
Fig. 2 is that freezing protectant is imported into the procedure chart in cell, and CPA is imported into the human body navel to cryo-conservation
Carrying substrates mescenchymal stem cell (human umbilical cord matrix mesenchymal stem cells, hUCM-
MSCs) in 6, illustrate process freezing protectant being imported into cell:
(1) CPA solution 5 is dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) and trehalose (trehalose)
Mixed liquor.The CPA solution of 3 kinds of various concentrations is configured, specifically, the CPA solution of each concentration all includes 0.5M's
Trehalose, but DMSO concentration difference, respectively 2M, 3M, 4M.Wherein, 3M is DMSO concentration needed for liquid nitrogen frozen.By 3
The CPA solution of kind various concentration is labeled as CPA#1, CPA#2, CPA#3.
(2) using dropper that the hUCM-MSCs culture solution (cell containing hUCM-MSCs 6) and the CPA#1 of 30ul of 30ul is successive
It imported into the hydrophobic surface area of SAW device.SAW device is driven using the alternating signal that power is 28dBm,
Under the driving of Rayleigh sound wave, CPA#1 starts to mix with hUCM-MSCs culture solution, and CPA#1 is gradually diffused into hUCM-MSCs
In cell wall, the aqueous solution in hUCM-MSCs cell 6 oozes out, and the volume of hUCM-MSCs cell 6 starts to reduce.In about 10s sound
Under the action of causing miniflow, the CPA concentration of intraor extracellular reaches balance, and the first step for completing CPA imports.
(3) on the basis of step (2), the CPA#2 of 30ul is imported by SAW device hydrophobic surface using dropper
Region, treated that hUCM-MSCs cell suspending liquid mixes with step (2).The driving power and frequency of SAW device are protected
Hold constant, under the driving of Rayleigh sound wave, CPA#2 starts to mix with hUCM-MSCs suspension, and CPA#2 is further spread
Into hUCM-MSCs cell wall, the aqueous solution in hUCM-MSCs cell 6 continues to ooze out, and the volume of hUCM-MSCs cell 6 is into one
Step reduces.Under the action of about 10s sound causes miniflow, the CPA concentration of intraor extracellular reaches balance, and the second step for completing CPA imports.
(4) on the basis of step (3), the CPA#3 of 30ul is imported by hydrophobic surface area and step (3) using dropper
HUCM-MSCs cell suspending liquid mixing that treated.The driving power and frequency of SAW device remain unchanged,
Under the driving of Rayleigh sound wave, CPA#3 starts to mix with hUCM-MSCs suspension, and CPA#3 continues to diffuse into hUCM-MSCs
In cell wall, the aqueous solution in hUCM-MSCs cell 6 further oozes out, and the volume of hUCM-MSCs cell 6 further decreases.?
Under the action of about 10s sound causes miniflow, the CPA concentration of intraor extracellular reaches balance, and the third step for completing CPA imports.
So far, the three step steps for importing of CPA are fully completed.HUCM-MSCs cell suspending liquid is stood 10 minutes, by expanding
The volume for dissipating hUCM-MSCs cell 6 after balancing is restored to original size.Three peaks of the coordinate curve in Fig. 2 illustrate
The situation of change of hUCM-MSCs cell cell volume in the three step importing process of CPA.
As shown in figure 3, the process of the freezing protectant in removal cell includes:
(a) the CPA solution of 6 kinds of concentration is configured, the content of DMSO is respectively 0M, 1M, 1.5M, 2M, 2.5M, 3M,
The content of trehalose is 0.5M, is respectively labeled as CPA0#, CPA1#, CPA2#, CPA3#, CPA4#, CPA5#.
(b) the hUCM-MSCs cell suspending liquid of CPA to be removed is imported into the hydrophobic surface area of SAW resonator
Domain, then the CPA#5 of 30ul is imported, the driving power and frequency of SAW device remain unchanged, in the drive of Rayleigh miniflow
Under dynamic, CPA#5 starts to mix with hUCM-MSCs cell suspending liquid, since the CPA concentration in hUCM-MSCs cell 6 is than extracellular
Height, aqueous solution are gradually diffused into hUCM-MSCs cell wall, and the CPA in cell gradually oozes out, the volume of hUCM-MSCs cell 6
Start to increase.SAW device works after 10s, completes the first step removal of CPA.
(c) on the basis of step (b), treated that hUCM-MSCs is outstanding in hydrophobic table and step (b) by drop 30ul CPA#4
Supernatant liquid mixing.The driving power and frequency of SAW device remain unchanged, under the driving of Rayleigh miniflow, CPA#4 with
6 culture solution of hUCM-MSCs cell starts to mix, and aqueous solution continues to diffuse into hUCM-MSCs cell wall, hUCM-MSCs cell 6
In CPA further ooze out, 6 volume of hUCM-MSCs cell further increases.SAW device works after 10s, completes CPA
Second step removal.
(d) on the basis of step (c), according to the process of step (c), by CPA3# and step (c) treated hUCM-
The mixing of MSCs suspension completes the third step removal of CPA with the CPA in diluting cells.
(e) on the basis of step (d), according to the process of step (c), by CPA2# and step (d) treated hUCM-
The mixing of MSCs suspension completes the 4th step removal of CPA with the CPA in diluting cells.
(f) on the basis of step (e), according to the process of step (c), by CPA1# and step (e) treated hUCM-
The mixing of MSCs suspension completes the 5th step removal of CPA with the CPA in diluting cells.
(g) on the basis of step (f), according to the process of step (c), by CPA0# and step (f) treated hUCM-
The mixing of MSCs suspension completes the 6th step removal of CPA with the CPA in diluting cells.
When step (a)~step (g) is completed, the CPA in hUCM-MSCs cell 6 is diluted to almost 0, so far thinks
CPA is completely removed.HUCM-MSCs cell suspending liquid is stood 10 minutes, until cell volume is restored to original size.Fig. 3
In 6 peaks of coordinate curve illustrate the variation feelings of hUCM-MSCs cell cell volume in the six step removal process of CPA
Condition.
The present embodiment utilizes the miniflow actuation effect using sound surface device, realizes the Quick uniform of CPA and cell solution
Mixing realizes the displacement of CPA and water intracellular by the concentration difference of CPA inside and outside cell wall.This method is realized by micro-fluidic device
Being sufficiently mixed for CPA and cell solution, reduces traditional manual mode of operation mechanical damage caused by cell.It is dense by CPA
The multistep for spending incremental (successively decreasing) imports (removal) method and mixes the CPA of various concentration with cell solution, can reduce in each step
CPA concentration gradient inside and outside cell wall reduces osmotic shock, preferably saves the form and integrality of cell, improves cell
Survival rate.
Further, it is also possible to which adjustment CPA solution imports (removal) by the driving power for accurately controlling SAW device
Speed obtains best CPA and imports (removal) effect, improves the survival rate of cell.It is done by the surface to SAW device thin
Water process is able to achieve the cell mobile mixing more smooth on its surface, further decreases the mechanical damage caused by cell.?
It is cooling using liquid nitrogen on the basis of this, transformation of the cell from liquid phase to glassy state may be implemented, no intracellular and extracellular crystallization occurs.
Embodiment 3
Such as Fig. 4 and Fig. 5, SAW device provided in this embodiment includes 128 ° of Y-cut LiNbO3Monocrystalline piezoelectric substrate
1, graphical interdigital transducer 2, interdigital transducer 9, interdigital transducer of the uniform deposition in lithium niobate monocrystal Piezoelectric Substrates 1
10, interdigital transducer 11.Oxygen plasma treatment first is carried out to the central crossbar region of 4 interdigital transducers, is then handed at center
It pitches one layer of silane of regional evapotranspiration (1H, 1,2h, 2H-perflurodecyltriethoxyl), to form hydrophobic surface 3.Miniflow
Pipeline 8 is between interdigital ring energy device 9 and 10, and microchannel 7 is between interdigital transducer 2 and interdigital transducer 11.It will be thin
Cytosol (freezing before or thaw after) is imported from microchannel 7,7 the right and left of microchannel interdigital transducer 2 and interdigital change
The sound of energy device 11 causes under miniflow driving, and cell liquid will flow to hydrophobic surface area.CPA solution 5 is imported from microchannel 8,
In the case where the sound of the interdigital transducer 9 and 10 of 8 the right and left of microchannel causes miniflow driving, CPA solution 5 will flow to hydrophobic surface
Region is mixed with cell liquid 6.
Freezing protectant importing minimizing technology process is carried out using the SAW device and embodiment 2 is identical, herein not
It repeats again.
Embodiment 4
A kind of Cryopreservation of cell is present embodiments provided, the Cryopreservation of the cell mainly includes following
Step:
Step 1, freezing protectant is imported into cell；
Step 2, liquid nitrogen frozen is carried out to the cell for having imported freezing protectant；
Step 3, water-bath defrosting is carried out to the cell of liquid nitrogen frozen；
Step 4, the freezing protectant in cell water-bath to be thawed removes.
Wherein, in step 1, by freezing protectant imported into the detailed process in cell such as the step (1) in embodiment 2~
After the completion of step (4), step (1) and step (4), cell suspending liquid is stood 10 minutes, the body of cell after diffusion balance
Product is restored to original size.
As shown in fig. 6, hUCM-MSCs cell suspending liquid is pipetted from hydrophobic surface to test tube using pipette in step 2
In, then hUCM-MSCs cell suspending liquid is transferred in suction pipe using syringe, and be placed on liquid nitrogen (LN2) in carry out it is fast
Fast vitrificated cryopreserration；
In step 3, when needing using hUCM-MSCs cell 6, suction pipe is removed from liquid nitrogen, and is placed on 37 DEG C of water
It thaws in bath 10.
In step 4, step in the detailed process such as embodiment 2 of the freezing protectant removal in cell that water-bath is thawed
(a)~step (g), when step (a)~step (g) is completed, intracellular CPA is diluted to almost 0, so far thinks CPA quilt
It completely removes.Cell suspending liquid is stood 10 minutes, until cell volume is restored to original size.
Compared with traditional manual manipulation mode, freezing protectant importing minimizing technology small, machine with osmotic shock
Tool damages low feature, so that cell integrity and form keep good, cell survival rate is greatly improved, can be to avoid artificial behaviour
It makes mistakes/biological cross contamination, the cryo-conservation etc. suitable for few cells.
Technical solution of the present invention and beneficial effect is described in detail in above-described specific embodiment, Ying Li
Solution is not intended to restrict the invention the foregoing is merely presently most preferred embodiment of the invention, all in principle model of the invention
Interior done any modification, supplementary, and equivalent replacement etc. are enclosed, should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of cause the freezing protectant of miniflow to import removal micro-system based on sound, which is characterized in that including SAW device,
Hydrophobic treatment is carried out to the part of the surface of SAW device, forms hydrophobic surface area, is completed in the hydrophobic surface area cold
Freeze the displacement of protective agent and water intracellular, realizes and freezing protectant is imported into cell and/or removes freezing protectant from cell.
2. causing the freezing protectant of miniflow to import removal micro-system based on sound as described in claim 1, which is characterized in that described
SAW device includes Piezoelectric Substrates, at least one interdigital transducer, wherein at least one interdigital transducer preparation exists
The piezoelectric substrate surface.
3. causing the freezing protectant of miniflow to import removal micro-system based on sound as claimed in claim 1 or 2, which is characterized in that
The driving microwave power of the SAW device is lower than+34dBm；The working frequency of the SAW device be 10~
100MHz。
4. causing the freezing protectant of miniflow to import removal micro-system based on sound as claimed in claim 1 or 2, which is characterized in that
The surface temperature when SAW device works is up to 37 DEG C.
5. a kind of cause the freezing protectant of miniflow to import minimizing technology based on sound, comprising the following steps:
(1) the described in any item freezing protectants of the Claims 1 to 4 are provided and import removal micro-system, the freezing protection
It includes SAW device that agent, which imports removal micro-system,；
(2) freezing protectant and cell culture fluid containing cell are imported by the sound table using dropper or micro-fluidic device
Hydrophobic surface area on surface wave device；
(3) SAW device described in the power drive lower than+34dBm, the SAW device generate surface acoustic wave,
Under the driving of the surface acoustic wave, the freezing protectant is uniformly mixed with the cell culture fluid, and outer freezing is anti-in the cell
It protects under diffusion caused by the concentration difference of agent, completes the displacement of freezing protectant and water intracellular, freezing protectant is led in realization
Enter cell or removes freezing protectant from cell.
6. causing the freezing protectant of miniflow to import minimizing technology based on sound as claimed in claim 5, which is characterized in that step
(3) in, SAW device described in control+27dBm~+34dBm power drive.
7. a kind of Cryopreservation of cell, comprising the following steps:
(1) minimizing technology being imported using the freezing protectant as described in claim 5 and 6, freezing protectant is imported into cell
In；
(2) liquid nitrogen frozen is carried out to the cell for having imported freezing protectant；
(3) water-bath defrosting is carried out to the cell of liquid nitrogen frozen；
(4) it is imported using the freezing protectant as described in claim 5 and 6 at the cell that minimizing technology thaws water-bath
Reason removes the freezing protectant in cell.
8. the Cryopreservation of cell as claimed in claim 7, which is characterized in that step (1) specifically includes:
(1-1) configuration has the freezing protectant of at least two kinds of concentration of concentration difference；
(1-2) imports the freezing protectant of minimum concentration and cell culture fluid containing cell by dropper or micro-fluidic device
To the hydrophobic surface area of the SAW device, the SAW device described in the power drive lower than+34dBm, in sound
Under the driving of surface wave, the freezing protectant of minimum concentration is uniformly mixed with cell culture fluid, causes the work of miniflow in 8~12s sound
Under, the freezing protectant concentration of intraor extracellular reaches balance, and the freezing protectant of minimum concentration is imported into the cell by realization；
(1-3) from low to high, using the method for step (1-2), successively imports the freezing protectant of various concentration according to concentration
To intracellular, until intracellular freezing protectant reaches aimed concn.
9. the Cryopreservation of cell as claimed in claim 7, which is characterized in that step (4) specifically includes:
(4-1) configuration has the freezing protectant of at least three kinds of concentration of concentration difference；
(4-2) imports the cell culture fluid that the freezing protectant of maximum concentration and water-bath thaw by dropper or micro-fluidic device
To the hydrophobic surface area of the SAW device, the SAW device described in the power drive lower than+34dBm, in sound
Under the driving of surface wave, the freezing protectant of maximum concentration is uniformly mixed with cell culture fluid, causes the work of miniflow in 8~12s sound
Under, the freezing protectant concentration of intraor extracellular reaches balance, with the freezing protectant in diluting cells；
(4-3) from high to low, using the method for step (4-2), successively utilizes the freezing protectant of various concentration dilute according to concentration
Intracellular freezing protectant is released, is completely removed until by intracellular freezing protectant.
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