CN117467601A - Exosome separation and purification method and system - Google Patents
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Abstract
The invention relates to an exosome separation and purification method and system, wherein the exosome separation and purification method comprises the following steps: obtaining tissue of exosomes to be extracted, separating cells containing exosomes from the tissue, culturing and amplifying in a culture medium, and collecting supernatant after cell culture as original exosome feed liquid; clarifying and filtering the original exosome feed liquid; carrying out tangential flow filtration on the feed liquid subjected to clarification filtration; purifying the feed solution subjected to tangential flow filtration in one or more combinations selected from the group consisting of: in the first mode, anion chromatography purification is carried out, and eluent is reserved; performing cation chromatography purification, and reserving flow-through liquid; step three, purifying by a size exclusion method, and reserving flow-through liquid; and (3) sterilizing the purified feed liquid. The invention can realize the purification of exosomes with different volumes and meet the quantity and purity of exosomes required clinically.
Description
Technical Field
The invention relates to the technical field of exosome purification, in particular to a separation and purification method and a separation and purification system for exosome.
Background
Exosomes (exosomes) are nanoscale vesicles 40-150 nm in diameter released outside cells during environmental stimulation or cell activation, mostly contain tubulin, actin, heat shock proteins, CD9, CD63 and the like, and also carry a large number of proteins derived from blast cells, the nucleic acids of which are mainly messenger RNAs and micrornas, and can regulate the expression of genetic information. The exosome can pass through biological barrier, carrier protein and nucleic acid to target cell, participate in immune response, antigen presentation, cell migration, cell differentiation, tumor invasion and other processes, and may be used as nano carrier to load gene or medicine to target organ.
The existing methods for separating and purifying exosomes include the following methods: (1) ultracentrifugation: when the mixture is subjected to centrifugal forces, the particulate components of the mixture will settle according to their density, size and shape. Ultracentrifugation is a centrifugal force of up to 10 6 g, centrifugal process. (2) size-screening-based separation techniques: ultrafiltration and size exclusion chromatography. The ultrafiltration method can achieve the aim of separation and purification through an ultrafiltration membrane for intercepting different relative molecular masses. Size exclusion chromatography, also known as size exclusion chromatography, space exclusion chromatography, is a method of separating solutes produced from the relative relationship between the size of the pore of a gel and the size of the molecules of the components to be separated, using the uniqueness of a porous gel stationary phase. (3) immunoaffinity capture-based separation techniques: immunoaffinity methods are based on the specific binding between antibodies and membrane proteins of exosomes to isolate exosomes. (4) separation technique of polymer precipitate: the polymer precipitation technology is that hydrophilic polymer interacts with hydrophilic bond of exosome in the sample to form hydrophobic micro environment around exosome, so as to form precipitate and extract exosome. (5) microfluidic-based separation techniques: the micro-fluidic technology can control the fluid behavior in the micro-channel, and the main fluid behavior is laminar flow and liquid drops, so that the purposes of precisely controlling the shape, the particle size and the like of the liquid drops are achieved. (6) artificial antibody-based separation techniques: the artificial antibody is a novel synthetic material which is based on molecular recognition and can specifically bind with a target object by simulating antibody-antigen interaction in a natural immune system. The above-mentioned several existing methods have advantages and disadvantages, but in summary, these methods cannot meet the clinical requirements of the obtained exosomes in terms of morphology, purity, yield and bioactivity, often require multiple separations or mixed use of multiple methods, and have large problems in terms of purification efficiency, working procedure operation and exosome yieldThe questions are given. Therefore, there is a need for further development of exosome purification techniques that can meet clinical requirements, have high purification efficiency, simplified procedures, and high yields.
Disclosure of Invention
In view of the above, the present invention provides a separation and purification method and system for exosomes, which can obtain exosomes with high purity and large quantity, thereby meeting the application requirements of clinical research and optimizing the operation procedures to a certain extent.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for separation and purification of exosomes, comprising the steps of:
step 1, obtaining tissue of exosomes to be extracted, separating cells containing the exosomes from the tissue, culturing and amplifying in a culture medium, and collecting supernatant after cell culture as original exosome feed liquid;
step 2, clarifying and filtering the original exosome feed liquid;
step 3, carrying out tangential flow filtration on the feed liquid subjected to the clarification filtration treatment;
step 4, purifying the feed liquid subjected to tangential flow filtration, wherein the purification mode is selected from one or more of the following modes:
in the first mode, anion chromatography purification is carried out, and eluent is reserved;
performing cation chromatography purification, and reserving flow-through liquid;
step three, purifying by a size exclusion method, and reserving flow-through liquid;
and 5, sterilizing the purified feed liquid.
Further, in the step 2, the clarification filtration treatment adopts a deep layer filter, preferably at least one of a PDK11 deep layer filter, a PDH4 deep layer filter, a V100P deep layer filter and a Profile deep layer filter series, wherein the filtration treatment comprisesThe filtering speed of the process control liquid is 150-500L/m 2 /h。
Preferably, in the step 2, the clarification filtration treatment adopts a PDK11 deep filter, the interception pore diameter is 2-20 mu m, the average filtration speed of the liquid in the filtration treatment process is about 400LMH, and the end point pressure difference is 1.0-1.5bar.
Further, in the step 3, the tangential flow filtration treatment device is subjected to cyclic disinfection treatment for 30min by adopting alkali liquor before the tangential flow filtration treatment, and the purified water is used for flushing the system to be neutral.
Preferably, in the step 3, the tangential flow filtration treatment adopts a TFF ultrafiltration membrane bag or a hollow fiber column with a molecular weight of 100kDa to 500kDa to carry out ultrafiltration concentration liquid exchange, wherein the ultrafiltration concentration multiple is 4 to 25 times, and the washing filtration liquid exchange multiple is 5 to 50 times; the pH value of the buffer solution for washing and filtering liquid is 4.5-9.0.
Further preferably, the tangential flow filtration treatment device adopts a TFF ultrafiltration membrane package with the specification of 300kDa, and the control parameters of the tangential flow filtration treatment are as follows: ultrafiltration (UF) concentration ratio is 10-15 times; the liquid change rate of the washing and filtering (DF) is 10-30 times; the pH value of the buffer solution for washing and filtering liquid is 5.0-8.0.
Preferably, the buffer solution is selected from one of 0.9% physiological saline, phosphate buffer, tris-HCl.
Further, in the step 4, a Mustang Q chromatographic membrane is used for the anion chromatography treatment; the cation chromatography treatment adopts a Mustang S chromatographic membrane; the size exclusion treatment process uses Capto Core400 or Capto Core700 packing.
The anion chromatography treatment can also be performed by Capto Q, capto DEAE, capto Q imprees, Q Sepharose XL, nuvia HP-Q, nuvia-Q from BIO-RAD, merckEMD TMAE、/>EMD DEAE filler and domestic filler of the same type.
The cationic chromatographic treatment can also be carried out by adopting CaptoSP, captoSPimpRes, captoSPimPact filler of cityva company and domestic filler of the same type.
Preferably, the control parameters for the Mustang Q treatment with the anion chromatographic membrane are: the pH value of the system is 7.0-8.0, and the sample is diluted 1-10 times for loading; the control parameters for Mustang S treatment with cationic chromatographic membranes were: the pH value of the system is 3.5-7.0, and the sample is diluted 1-10 times for loading; the control parameters for size exclusion treatment with Capto Core700 or Capto Core400 filler were: the pH value of the buffer solution is 7.0-8.0, and the sample is loaded according to the range of 0.25-1.5 of the ratio (CV) of the sample loading volume to the filler volume.
Further, the sterilizing filter in the step 5 is a sterilizing filter with a interception pore size of 0.2 μm.
Preferably, in the step 5, the sterilization is performed by using an ECV filter.
In a second aspect, the present invention provides an exosome purification system comprising a pretreatment device, a clarification filtration device, a tangential flow filtration device, a purification device, and a sterilization device connected in sequence;
the pretreatment device is used for culturing and amplifying cells separated from tissues and collecting original exosome feed liquid;
the clarifying and filtering device is used for clarifying and filtering the exocrine body fluid;
the tangential flow filtration device is used for carrying out tangential flow filtration treatment on the clarified and filtered exotic body fluid;
the purification device is used for purifying the external body fluid subjected to tangential flow filtration treatment;
the degerming device is used for degerming the purified exocrine body fluid.
Preferably, the clarification filter device is a PDK11 depth filter.
Further, the tangential flow filtration device comprises an ultrafiltration unit and a wash filtration unit connected to each other.
Further, the tangential flow filtration processing device employs a TFF membrane package of 300kDa gauge.
Further, the purification device comprises an ion chromatography device and/or a size exclusion device, wherein the ion chromatography device is a Mustang Q anion exchange chromatography device and/or a Mustang S cation exchange chromatography device; the size exclusion device is a Capto Core700 or Capto Core400 size exclusion device.
Further, the sterilization device is an ECV filter.
The beneficial effects of the invention are as follows: the invention develops a purification method of clinical grade exosome process flow and quality standard, and meets the quantity and purity of exosomes required clinically. The invention can realize the purification of exosomes with different volumes (100 ml to 100L of original exosome feed liquid).
Drawings
FIG. 1 shows the filtration rate flux curves of two filters according to example 1 of the present invention, wherein a) shows the filtration rate flux curve of PDH4 and b) shows the filtration rate flux curve of PDK 11.
FIG. 2 shows different flow rate curves for the PDK11 filter according to example 1 of the present invention, wherein a) shows a filtration rate of 150LMH (same as b) in FIG. 1) and b) shows a filtration rate of 400LMH.
FIG. 3 shows the absorbance peaks of UV280 and UV260 at two pH values using a Mustang Q ion chromatography device in example 1 of the invention, wherein a) is pH7.0 and b) is pH8.0.
FIG. 4 is a schematic diagram showing the particle size distribution of the finally purified exosomes in example 2 of the present invention.
FIG. 5 is a schematic diagram showing the particle size distribution of the finally purified exosomes in example 3 of the present invention.
FIG. 6 is a schematic diagram showing the particle size distribution of the finally purified exosomes in example 4 of the present invention.
FIG. 7 is a schematic diagram showing the electron microscopic observation of the finally purified exosomes in example 2 of the present invention.
FIG. 8 is a schematic diagram showing the electron microscopic observation of the finally purified exosomes in example 3 of the present invention.
FIG. 9 is a schematic diagram showing the electron microscopic observation of the finally purified exosomes in example 4 of the present invention.
FIG. 10 shows the protein markers (CD 63, CD 81) of the final purified exosomes in example 2 of the present invention.
FIG. 11 shows the protein markers (CD 63, CD 81) of the final purified exosomes in example 3 of the present invention.
FIG. 12 shows protein markers of the final purified exosomes in example 4 of the present invention.
FIG. 13 is a schematic diagram showing the structure of an exosome purification system according to the present invention, wherein 1, pretreatment device, 2, clarification filtration device, 3, tangential flow filtration device, 3-1, ultrafiltration unit, 3-2, washing filtration unit, 4, purification device, 5, and sterilization device.
Detailed Description
The filter membrane type adopted in the embodiment of the invention is PDK11 and PDH4 deep filter which are purchased from Cytiva company.
TFF devices employed in embodiments of the present invention were purchased from Cytiva corporation.
The Mustang Q XT Acrodisc, mustang S XT Acrodisc ion exchange chromatography apparatus used in the examples of the present invention was purchased from Cytiva corporation.
The EKV filters used in the examples of the present invention were purchased from Cytiva.
Capto Core700 and Capto Core400 size exclusion devices used in embodiments of the invention were purchased from Cytiva corporation.
The sterile ECV filter used in the examples of the present invention was purchased from Cytiva.
In the description of the present invention, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides an exosome purification method, which comprises the following steps:
step 1, obtaining tissue of exosomes to be extracted, separating cells containing the exosomes from the tissue, culturing and amplifying in a culture medium, and collecting supernatant after cell culture as original exosome feed liquid;
step 2, clarifying and filtering the original exosome feed liquid;
step 3, carrying out tangential flow filtration on the feed liquid subjected to the clarification filtration treatment;
step 4, purifying the feed liquid subjected to tangential flow filtration, wherein the purification mode is selected from one or more of the following modes:
in the first mode, anion chromatography purification is carried out, and eluent is reserved;
performing cation chromatography purification, and reserving flow-through liquid;
step three, purifying by a size exclusion method, and reserving flow-through liquid;
and 5, sterilizing the purified feed liquid.
In the above step 1, the exosome-containing tissue includes umbilical cord tissue, menstrual blood, endometrium, amniotic membrane, bone marrow, dental pulp, fat, etc.; after mesenchymal stem cells containing exosomes are separated and extracted from the tissues, the stem cells are subjected to expansion culture to reach the generation P5, the expansion culture process can be realized by adopting the conventional technology at present, and when the cell fusion degree reaches 70-90% or more, the cell morphology is normal and the adherent growth state is good.
In the step 2, the liquid filtering speed is controlled to be 150-500L/m in the clarification and filtration treatment process 2 /h。
In the step 3, the tangential flow filtration treatment device is circularly disinfected for 30min by alkali liquor before the tangential flow filtration treatment, and the system is flushed to be neutral by purified water.
Preferred control parameters for the tangential flow filtration process are: the ultrafiltration concentration multiple is 4-25 times, and the washing filtration liquid exchange multiple is 5-50 times; the pH value of the buffer solution for washing and filtering liquid is 4.5-9.0. Such as: buffer solutions that may be used are: 0.9% physiological saline; phosphate buffer at pH 7.4; a mixed solution of 25mM Tris-HCl and 200mM NaCl, pH 7.0;10mM Tris-HCl solution, pH 8.0; a mixed solution of 20mM Tris-HCl and 150mM NaCl, pH7.5; preferably, it is: a mixed solution of 20mM Tris-HCl and 150mM NaCl, pH7.5.
Wherein, the concentration multiple refers to the ratio of the liquid volumes before and after concentration; the multiple of the change of the liquid is the ratio of the volume of the buffer solution to the volume of the concentrated liquid.
In the above step 4, if a plurality of modes are combined, the first mode, the second mode and the third mode can be arranged and used in any order, for example: each treatment can be sequentially carried out for 1 time or each treatment is carried out for a plurality of times or is circulated for a plurality of times according to the sequence of the first mode, the second mode and the third mode, and vice versa; or, each treatment is sequentially carried out for 1 time or each treatment is carried out for a plurality of times or is circulated for a plurality of times according to the sequence of the first mode and the second mode, and vice versa; or, each treatment is sequentially carried out for 1 time or each treatment is carried out for a plurality of times or is circulated for a plurality of times according to the sequence of the first mode and the third mode, and vice versa; or sequentially carrying out treatment for 1 time or treatment for multiple times or circulation for multiple times according to the sequence of the second mode and the third mode; or mode two to mode one, or vice versa; etc.
Among these various combinations described above, preferred modes are: 1 treatment per mode + 1 treatment per mode three.
The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.
Example 1
Screening process of exosome separation and purification control parameters:
(1) Deep filter screening
Umbilical cord tissue is donated from a volunteer in gynaecology and obstetrics hospitals in Hangzhou, the source of the umbilical cord tissue accords with the evaluation of ethics committee of gynaecology and obstetrics hospitals in Hangzhou, and raw exosome feed liquid extracted from stem cells from the umbilical cord tissue (an extraction method is shown in example 2) is thawed overnight at 4 ℃ and then mixed uniformly. Clarification was performed in a constant flow manner using depth filters PDK11 and PDH4, the results of which are shown in FIGS. 1 and 2.
At a flow rate of 150LMH, the PDH4 filter had a filtration capacity of 331.9L/m 2 The filtration capacity of the PDK11 filter is 492.2L/m 2 . From the results, the PDK11 filter loading was 48% higher than the PDH4 filter loading, so a higher loading PDK11 filter was selected.
After setting the flow rate of the PDK11 filter to 400LMH, the filtration capacity became 366.5L/m 2 . Compared with the flow rate of 150LMH, the method shortens the process time by 68 percent, but feeds the sample moreThe flow rate causes the depth filter to reach the pressure endpoint faster, reducing the filter loading by 25.5%.
(2) Screening of purification modes
(2-1) treatment with Mustang Q anion chromatography alone
Clarifying and filtering by adopting a PDK11 filter, and then carrying out tangential flow filtration on the clarified and filtered feed liquid by adopting a 100kDa TFF ultrafiltration membrane package, wherein the control parameters of the tangential flow filtration are as follows: ultrafiltration (UF) concentration ratio was 20 times; the rate of liquid change of the washing and filtering (DF) is 7 times, the pH value of the buffer solution for liquid change of the washing and filtering is 7.0 and 8.0, and the purification results under the two pH values are examined.
As shown in FIG. 3, the pH7.0 chromatography was severe in the flow-through phenomenon, the recovery rate was low, the flow-through phenomenon was small after pH8.0 was used, and the elution recovery rate was increased.
The BSA (bovine serum albumin) and NTA (nanoparticle tracking analysis) detection conditions at each of the two pH stages are shown in table 1:
TABLE 1BSA and NTA detection results
In combination with the BSA removal and target particle recovery at each stage, if the purification is performed by an independent anion chromatography, the recovery rate after the chromatographic elution of the system with pH8.0 is high, and the flow through phenomenon is lower than pH7.0, so the system with pH8.0 is considered to be adopted at the chromatographic stage.
(2-2) treatment with different chromatography combinations
The original exosome feed liquid extracted from stem cells of menstrual blood tissue source (the extraction method is shown in example 3) is thawed overnight at 4 ℃ and then mixed uniformly. Clarifying and filtering by adopting a PDK11 filter, and then carrying out tangential flow filtration on the clarified and filtered feed liquid by adopting a 300kDa TFF ultrafiltration membrane package, wherein the control parameters of the tangential flow filtration are as follows: ultrafiltration (UF) concentration ratio is 10 times; the liquid exchange rate of the washing and filtering (DF) is 10 times, the pH value of the buffer solution for washing and filtering liquid exchange is 8.0, the liquid after washing and filtering treatment is treated by adopting different purification chromatography modes, the target product is collected, and the purification results of different purification combination modes are examined.
TABLE 2BSA and nanofluidic particle detection results
Clearly, good BSA removal was achieved for all three purification modes, but the recovery of purification 1 was slightly higher than that of purification 2 and 3, so that purification 1 was considered as the relatively preferred treatment mode for the present application.
(3) Screening of a sterilizing device
Clarifying and filtering by adopting a PDK11 filter, and then carrying out tangential flow filtration on the clarified and filtered feed liquid by adopting a 300kDa TFF ultrafiltration membrane package, wherein the control parameters of the tangential flow filtration are as follows: ultrafiltration (UF) concentration ratio is 12 times; the liquid exchange rate of the washing and filtering (DF) is 10 times, the pH value of the buffer solution for washing and filtering liquid exchange is 7.5, the liquid after washing and filtering treatment is treated by Capto Core700 chromatography, the eluent is collected, the eluent is sterilized by using different types of 0.2 mu m sterilizing filters, and the influence of different types of sterilizing filters on the recovery rate of exosomes is examined.
The nanoparticle tracking analysis detection of the filtrate after sterilization by two different types of sterilization filters is shown in table 3:
TABLE 3 nanometer flow particle detection results
| Step (a) | Filter model | Particle count (Particles) | Recovery rate |
| Degerming | EKV | 1.2E+10 | 23.1％ |
| Degerming | ECV | 3.0E+11 | 73％ |
The recovery rate of the ECV sterilizing filter is obviously higher than that of the EKV sterilizing filter according to the recovery condition of target particles, so the ECV sterilizing filter is considered to be used in the sterilizing stage.
Example 2
The tissue adopted in this example is umbilical cord tissue, the umbilical cord tissue is donated from a volunteer of gynaecology and obstetrics hospital in Hangzhou, and the umbilical cord tissue is from a source conforming to the evaluation of ethics committee of gynaecology and obstetrics hospital in Hangzhou, the treatment method comprises separating mesenchymal stem cells by tissue adherence method, culturing and amplifying in a culture medium, the culture medium is composed of DMEM/F12 and UltroserTM G serum substitute, wherein the volume ratio of UltroserTM G serum substitute in the basic culture medium is 2%, the culture medium is prepared from 5% CO at 37 DEG C 2 Observing and changing liquid every 2-3 days, carrying out pancreatin digestion passage when the fusion degree reaches 70-90%, wherein the pancreatin is 0.25% and 0.125% of Trypsin-EDTA, carrying out passage amplification culture on the cells until the generation P5, and collecting supernatant after cell culture as original exosome liquid when the fusion degree reaches 70-90%.
The experimental process comprises the following steps:
1. clarifying and filtering
Mixing 2L of the upstream feed liquid uniformly, clarifying and filtering by using a deep filter model SC050PDK11 to remove dead cells and cell fragments, and finishing a clarification process in a constant flow mode with an average flow rate of 400LMH, wherein the pressure end point is 1.0-1.5bar, thus obtaining clarified feed liquid.
2. TFF (tangential flow filtration): UF (ultra filtration)/DF (diafiltration) tangential flow filtration
The clarified feed liquid is subjected to ultrafiltration concentration of clarified liquid by using a 100kDa TFF ultrafiltration membrane to cover OS100T02, and the inlet flow rate is controlled to be about 4.5-5.0L/min/m 2 The transmembrane pressure was controlled at about 1.0bar and the concentration was 10-fold. After the concentration, the chromatographic equilibrium buffer solution 10mM Tris-HCl, pH8.0 is used for continuous liquid exchange, and the inlet flow rate is controlled to be about 4.0-5.0L/min/m 2 The transmembrane pressure is controlled to be about 1.0-1.45 bar, and the liquid exchange multiple is 12 times.
3. Ion chromatography treatment
And (3) carrying out membrane chromatography on the feed liquid subjected to ultrafiltration washing by using an anion chromatographic membrane with the model of XT5MSTGQPM 6.
1) The anion chromatographic membrane of XT5MSTGQPM6 was attached to the AKTA Avant 150 chromatography system.
2) Rinsing the membrane chromatographic filter with 1M NaOH at a flow rate of 25mL/min, and then rinsing the membrane chromatographic filter with deionized water;
3) The membrane filter was rinsed with 10mM Tris-HCl,1M NaCl,pH8.0, flow rate 25ml/min;
4) Buffer equilibration was performed with 10mM Tris-HCl, pH8.0, flushing the membrane chromatographic filter to baseline plateau at a flow rate of 25ml/min;
5) Concentrating and washing the TFF, and loading the sample at a flow rate of 15mL/min;
6) The membrane filter was rinsed with 10mM Tris-HCl, pH8.0, to baseline equilibrium at a flow rate of 15ml/min;
7) Eluting with 10mM Tris-HCl and 1M NaCl at a gradient of 0-100% B, and collecting a sample when an ultraviolet peak UV280 appears at a flow rate of 15ml/min;
8) Washing the membrane chromatographic filter with 1M NaOH after the step 7 is finished;
9) And (3) preserving: the membrane filter was rinsed with 0.1M NaOH,1M NaCl.
4. Degerming and filtering
And (3) using an ECV filter with the model, intercepting the material liquid with the aperture of 0.2 mu m, finishing the sterilization process on the material liquid after anion membrane chromatography in a constant flow mode with the average flow rate of about 500LMH, wherein the pressure end point is 2bar, and collecting a sample to obtain the purified exosome final product.
Example 3
The tissue used in this example was menstrual blood (including endometrial tissue), the treatment method was mixing the collected menstrual blood, filtering the mixture with a blood transfusion filter screen to remove mucus and lumpy substances, mixing the cell filtrate, taking a proper amount of WBCs, mixing the filtrate, centrifuging to remove the supernatant, resuspending the cell pellet with Phosphate Buffer (PBS) pH containing vancomycin hydrochloride antibiotic (anti-PBS), centrifuging to wash for 2 times, and setting the centrifugation parameters to 300g, 6min. Adding the cells containing anti-PBS to the suspension, and adjusting WBC concentration to (0.20-1.00) x 10 7 Performing density gradient centrifugation and purification on cells/ml according to the volume ratio of 2:1, setting the centrifugation parameters to 700g and 30min, sucking the upper layer solution, sucking the tunica albuginea layer cells (mononuclear cells), adding the anti-PBS, uniformly mixing, and centrifuging for 800g and 6min. Removing supernatant, adding cell sediment containing anti-PBS, centrifuging and cleaning for 300g and 6min, mixing with cell sediment containing anti-PBS, mixing into 1 tube, centrifuging for 300g and 6min. Resuspension of the cell pellet with M4 complete medium containing vancomycin hydrochloride antibiotic to obtain primary cells, inoculating several bottles of T75 bottles, density of 60.0X10 6 cells/flask, culture and amplification in a medium comprising DMEM/F12 and UltroserTM G serum substitute, wherein UltroserTM G serum substitute is 2% by volume in the basal medium and cells are cultured at 37deg.C and 5% CO 2 And (3) culturing cells in an incubator, observing and changing the liquid every 2-3 days, when the fusion degree reaches 70-90%, using 1.2mg of recombinant trypsin solution for digestion, carrying out passage amplification culture on the cells until the generation of P5, and when the fusion degree reaches 70-90%, collecting supernatant after the cell culture as original exosome liquid.
The experimental process comprises the following steps:
1. clarifying and filtering
Mixing 2L of upstream feed liquid, clarifying and filtering with a deep filter model SC050PDK11 to remove dead cells and cell fragments, and completing a clarification process in a constant flow mode with an average flow rate of 400LMH at a pressure end point of 1.0-1.5bar to obtain clarified feed liquid.
2. TFF (tangential flow filtration): UF (ultra filtration)/DF (diafiltration) tangential flow filtration
The clarified feed liquid is subjected to ultrafiltration concentration of clarified liquid by using a 300kDa TFF ultrafiltration membrane to wrap OS300T12, and the inlet flow rate is controlled to be about 3-4L/min/m 2 Controlling the transmembrane pressure to be about 0.2-0.3 bar, and concentrating by 12 times. After the concentration, the solution is continuously exchanged by using 20mM Tris-HCl,150mM NaCl,pH7.5 buffer solution, and the inlet flow rate is controlled to be about 3-4L/min/m 2 The transmembrane pressure is about 0.18-0.26 bar, and the liquid exchange multiple is 10 times.
3. Size exclusion treatment
And (3) performing size exclusion treatment on the feed liquid subjected to ultrafiltration washing and filtering by using Capto Core700 filler.
1) The chromatography column loaded with 100ml Capto Core700 packing was connected to an AKTA Avant 150 chromatography system.
2) Flushing the chromatographic column with deionized water;
3) Washing the chromatographic column with 1M NaOH at a flow rate of 5ml/min;
4) Buffer equilibration: washing the chromatographic column with 20mM Tris-HCl,150mM NaCl,pH7.5 to baseline plateau at a flow rate of 10ml/min;
5) Loading: 75ml (0.75 CV) of TFF concentrated and washed filtered sample is loaded at a flow rate of 5ml/min, and a flow-through mode is adopted to collect a flow-through main peak of UV280 combined with UV 216;
6) Washing the chromatographic column with 20mM Tris-HCl,150mM NaCl,pH7.5 to baseline plateau at a flow rate of 10ml/min;
7) Eluting with 1M NaOH at a flow rate of 10ml/min, and collecting no ultraviolet peak;
8) Washing the chromatographic column with 20mM Tris-HCl,150mM NaCl,pH7.5 to baseline plateau at a flow rate of 10ml/min;
9) And (3) preserving: the column was washed with 20% ethanol solution.
4. Degerming and filtering
And (3) using an ECV filter with the model, intercepting the material liquid with the aperture of 0.2 mu m, finishing the sterilization process on the material liquid after anion membrane chromatography in a constant flow mode with the average flow rate of about 500LMH, wherein the pressure end point is 2bar, and collecting a sample to obtain the purified exosome final product.
Example 4
The tissue used in this example was menstrual blood (including endometrial tissue), the treatment method was mixing the collected menstrual blood, filtering the mixture with a blood transfusion filter screen to remove mucus and lumpy substances, mixing the cell filtrate, taking a proper amount of WBCs, mixing the filtrate, centrifuging to remove the supernatant, resuspending the cell pellet with Phosphate Buffer (PBS) pH containing vancomycin hydrochloride antibiotic (anti-PBS), centrifuging to wash for 2 times, and setting the centrifugation parameters to 300g, 6min. Adding the cells containing anti-PBS to the suspension, and adjusting WBC concentration to (0.20-1.00) x 10 7 Performing density gradient centrifugation and purification on cells/ml according to the volume ratio of 2:1, setting the centrifugation parameters to 700g and 30min, sucking the upper layer solution, sucking the tunica albuginea layer cells (mononuclear cells), adding the anti-PBS, uniformly mixing, and centrifuging for 800g and 6min. Removing supernatant, adding cell sediment containing anti-PBS, centrifuging and cleaning for 300g and 6min, mixing with cell sediment containing anti-PBS, mixing into 1 tube, centrifuging for 300g and 6min. Resuspension of the cell pellet with M4 complete medium containing vancomycin hydrochloride antibiotic to obtain primary cells, inoculating several bottles of T75 bottles, density of 60.0X10 6 cells/flask, culture and amplification in a medium comprising DMEM/F12 and UltroserTM G serum substitute, wherein UltroserTM G serum substitute is 2% by volume in the basal medium and cells are cultured at 37deg.C and 5% CO 2 And (3) culturing cells in an incubator, observing and changing the liquid every 2-3 days, when the fusion degree reaches 70-90%, using 1.2mg of recombinant trypsin solution for digestion, carrying out passage amplification culture on the cells until the generation of P5, and when the fusion degree reaches 70-90%, collecting supernatant after the cell culture as original exosome liquid.
The experimental process comprises the following steps:
1. clarifying and filtering
Mixing 2L of upstream feed liquid, clarifying and filtering with a deep filter model SC050PDK11 to remove dead cells and cell fragments, and completing a clarification process in a constant flow mode with an average flow rate of 400LMH at a pressure end point of 1.0-1.5bar to obtain clarified feed liquid.
2. TFF (tangential flow filtration): UF (ultra filtration)/DF (diafiltration) tangential flow filtration
The clarified feed liquid is subjected to ultrafiltration concentration of clarified liquid by using a 300kDa TFF ultrafiltration membrane to wrap OS300T12, and the inlet flow rate is controlled to be about 4-5L/min/m 2 Controlling the transmembrane pressure to be about 0.2-0.3 bar, and concentrating 11 times. After the concentration is finished, the solution is continuously changed by using 0.9 percent physiological saline, and the flow rate of a control port is about 4 to 5L/min/m 2 The transmembrane pressure was controlled at about 0.36bar and the fold of the change was 30 times.
3. Size exclusion treatment
And (3) performing size exclusion treatment on the feed liquid subjected to ultrafiltration washing and filtering by using Capto Core700 filler.
1) The chromatography column loaded with 100mlCapto Core700 packing was connected to an AKTA Avant 150 chromatography system.
2) Flushing the chromatographic column with deionized water;
3) Washing the chromatographic column with 1M NaOH at a flow rate of 5ml/min;
4) Buffer equilibration: flushing the chromatographic column with 0.9% physiological saline until the chromatographic column is stable to a base line, and the flow rate is 10ml/min;
5) Loading: 50ml (0.5 CV) of TFF concentrated and washed filtered sample is loaded at a flow rate of 5ml/min, and a flow-through mode is adopted to collect a flow-through main peak of UV280 combined with UV 216;
6) Flushing the chromatographic column with 0.9% physiological saline until the chromatographic column is stable to a base line, and the flow rate is 10ml/min;
7) Eluting with 1M NaOH at a flow rate of 10ml/min, and collecting no ultraviolet peak;
8) Washing the chromatographic column with 50mM Tris-HCl,150mM NaCl,pH7.5 to baseline plateau at a flow rate of 10ml/min;
9) And (3) preserving: the column was washed with 20% ethanol solution.
4. Degerming and filtering
And (3) using an ECV filter with the model, intercepting the material liquid with the aperture of 0.2 mu m, finishing the sterilization process on the material liquid after anion membrane chromatography in a constant flow mode with the average flow rate of about 500LMH, wherein the pressure end point is 2bar, and collecting a sample to obtain the purified exosome final product.
Example 5: detection of exosome samples
The exosome samples prepared in examples 2-4 were tested and analyzed, the exosome particle size distribution diagram is shown in fig. 4-6, the exosome electron microscope observation schematic diagram is shown in fig. 7-9, the protein markers are shown in fig. 10-12, and the plurality of column strips in fig. 10-12 are a plurality of batches.
The exosomes obtained in this example were identified for morphology, purity, yield and bioactivity as follows:
in the BSA detection conditions of the embodiments, the BSA clearance is up to 99%, the concentration of nano-flow detection particles is higher than 30%, and the recovery rate is higher than 30%. As shown in table 4:
TABLE 4 Table 4
Example 6
The present embodiment provides an exosome purification system, as shown in fig. 13, which includes a pretreatment device 1, a clarification filtration device 2, a tangential flow filtration device 3, a purification device 4, and a sterilization device 5, which are sequentially connected;
the pretreatment device 1 is used for culturing and amplifying cells separated from tissues and collecting original exosome feed liquid;
the clarifying and filtering device 2 is used for clarifying and filtering the exocrine body fluid;
the tangential flow filtration device 3 is used for carrying out tangential flow filtration treatment on the clarified and filtered exotic body fluid;
the purification device 4 is used for purifying the external body fluid subjected to tangential flow filtration treatment;
the sterilization device 5 is used for sterilizing the purified exocrine body fluid.
In this embodiment, the clarifying filter device 2 is a PDK11 depth filter.
In this embodiment, the tangential flow filtration device 3 comprises an ultrafiltration unit 3-1 and a wash filtration unit 3-2 connected to each other.
In this embodiment, the ultrafiltration unit 3-1 and the washing unit 3-2 of the tangential flow filtration treatment device 3 each employ a TFF membrane package having a specification of 300 kDa.
In this embodiment, the purification device 4 comprises an ion chromatography device and/or a size exclusion device, which is a Mustang Q anion exchange chromatography device and/or a Mustang S cation exchange chromatography device; the size exclusion device is a Core700 size exclusion device.
In this embodiment, the sterilization device 5 is an ECV filter.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. An exosome separation and purification method is characterized in that: comprising the following steps: the method comprises the following steps:
step 1, obtaining tissue of exosomes to be extracted, separating cells containing the exosomes from the tissue, culturing and amplifying in a culture medium, and collecting supernatant after cell culture as original exosome feed liquid;
step 2, clarifying and filtering the original exosome feed liquid;
step 3, carrying out tangential flow filtration on the feed liquid subjected to the clarification filtration treatment;
step 4, purifying the feed liquid subjected to tangential flow filtration, wherein the purification mode is selected from one or more of the following modes:
in the first mode, anion chromatography purification is carried out, and eluent is reserved;
performing cation chromatography purification, and reserving flow-through liquid;
step three, purifying by a size exclusion method, and reserving flow-through liquid;
and 5, sterilizing the purified feed liquid.
2. The method for separating and purifying exosomes according to claim 1, wherein: in the step 2, the clarifying filtration treatment adopts a deep layer filter, preferably at least one of a PDK11 deep layer filter, a PDH4 deep layer filter, a V100P deep layer filter and a Profile deep layer filter series, wherein the liquid filtration speed is controlled to be 150-500L/m in the filtration treatment process 2 /h。
3. The method for separating and purifying exosomes according to claim 1, wherein: in the step 3, alkali liquor is adopted to circularly disinfect the tangential flow filtration treatment device for 30min before the tangential flow filtration treatment, and a purified water flushing system is used to be neutral; preferably, the tangential flow filtration treatment adopts a TFF ultrafiltration membrane bag or a hollow fiber column of 100 kDa-500 kDa to carry out ultrafiltration concentration liquid exchange, wherein the ultrafiltration concentration multiple is 4-25 times, and the washing filtration liquid exchange multiple is 5-50 times; the pH value of the buffer solution for washing and filtering liquid is 4.5-9.0.
4. The method for separating and purifying exosomes according to claim 1, wherein: in the step 4, a Mustang Q chromatographic membrane is adopted for anion chromatography treatment; the cation chromatography treatment adopts a Mustang S chromatographic membrane; the size exclusion treatment process uses Capto Core400 or Capto Core700 packing.
5. The method for separating and purifying exosomes according to claim 4, wherein: the control parameters for the Mustang Q treatment with the anion chromatographic membrane were: the pH value of the system is 7.0-8.0, and the sample is diluted 1-10 times for loading; the control parameters for Mustang S treatment with cationic chromatographic membranes were: the pH of the system is 5.0-7.0, and the sample is diluted 1-10 times for loading; the control parameters for size exclusion treatment with Capto Core700 or Capto Core400 filler were: the pH value of the buffer solution is 7.0-8.0, and the sample is loaded according to the range of 0.25-1.5 of the loading volume and the filling volume ratio.
6. The method for separating and purifying exosomes according to claim 1, wherein: the sterilizing filter in the step 5 is a sterilizing filter with interception pore diameter of 0.2 μm.
7. An exosome purification system, characterized by: comprises a pretreatment device, a clarification and filtration device, a tangential flow filtration device, a purification device and a sterilization device which are connected in sequence;
the pretreatment device is used for culturing and amplifying cells separated from tissues and collecting original exosome feed liquid;
the clarifying and filtering device is used for clarifying and filtering the exocrine body fluid;
the tangential flow filtration device is used for carrying out tangential flow filtration treatment on the clarified and filtered exotic body fluid;
the purification device is used for purifying the external body fluid subjected to tangential flow filtration treatment;
the degerming device is used for degerming the purified exocrine body fluid.
8. An exosome purification system according to claim 7, wherein: the tangential flow filtration device comprises an ultrafiltration unit and a wash filtration unit connected to each other.
9. An exosome purification system according to claim 7, wherein: the purification device comprises an ion chromatography device and/or a size exclusion device, wherein the ion chromatography device is a Mustang Q anion exchange chromatography device and/or a Mustang S cation exchange chromatography device; the size exclusion device is a Capto Core700 or Capto Core400 size exclusion device.
10. An exosome purification system according to claim 7, wherein: the sterilization device is an ECV filter.
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