WO2014089838A1 - 离心动态过滤装置及利用其的细胞分离系统 - Google Patents

离心动态过滤装置及利用其的细胞分离系统 Download PDF

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Publication number
WO2014089838A1
WO2014089838A1 PCT/CN2012/086694 CN2012086694W WO2014089838A1 WO 2014089838 A1 WO2014089838 A1 WO 2014089838A1 CN 2012086694 W CN2012086694 W CN 2012086694W WO 2014089838 A1 WO2014089838 A1 WO 2014089838A1
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WO
WIPO (PCT)
Prior art keywords
membrane
filter
pipe
liquid
cell suspension
Prior art date
Application number
PCT/CN2012/086694
Other languages
English (en)
French (fr)
Inventor
郑翀
Original Assignee
Zheng Chong
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP12890047.9A priority Critical patent/EP2933326B1/en
Priority to MYPI2014003212A priority patent/MY171082A/en
Application filed by Zheng Chong filed Critical Zheng Chong
Priority to AU2012396668A priority patent/AU2012396668B2/en
Priority to KR1020147032756A priority patent/KR101728939B1/ko
Priority to SG11201408169YA priority patent/SG11201408169YA/en
Priority to RU2014144222/10A priority patent/RU2600871C2/ru
Priority to JP2015516411A priority patent/JP5925962B2/ja
Priority to CA2873077A priority patent/CA2873077C/en
Priority to ES12890047T priority patent/ES2874956T3/es
Priority to CN201280063057.5A priority patent/CN104363972B/zh
Priority to PCT/CN2012/086694 priority patent/WO2014089838A1/zh
Priority to PT128900479T priority patent/PT2933326T/pt
Priority to BR112014029935A priority patent/BR112014029935B1/pt
Publication of WO2014089838A1 publication Critical patent/WO2014089838A1/zh
Priority to US14/547,159 priority patent/US20150068959A1/en
Priority to US15/230,516 priority patent/US10258927B2/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C12M1/123—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means with flat plate filter elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D33/00—Filters with filtering elements which move during the filtering operation
    • B01D33/06—Filters with filtering elements which move during the filtering operation with rotary cylindrical filtering surfaces, e.g. hollow drums
    • B01D33/067—Construction of the filtering drums, e.g. mounting or sealing arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D33/00—Filters with filtering elements which move during the filtering operation
    • B01D33/80—Accessories
    • B01D33/804—Accessories integrally combined with devices for controlling the filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/16—Rotary, reciprocated or vibrated modules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/02—Rotation or turning

Definitions

  • the invention relates to the field of biological cell separation, in particular to a centrifugal dynamic filtering device and a cell separation system comprising the microporous membrane filter, by which a rapid cell separation method can be established.
  • Cell therapy One of the best ways to treat disease is to use living tissue to reconstruct and renew disease and aging tissue; this treatment is called "cell therapy.”
  • Cell therapy has been used for centuries and is widely used in various fields such as tumors, liver diseases, and scarring, and has broad prospects for development.
  • One of the objects of the present invention is to provide a centrifugal dynamic filtering device which is simple in structure, convenient in operation, capable of rapidly separating cells, and can be separated in a fully enclosed system to alleviate cell damage caused by the separation process and is less likely to cause cell contamination.
  • the whole process of separation can be fully automated control of the computer.
  • a centrifugal dynamic filtering device for living cell separation comprising: a rotating shaft, a rotating arm vertically connected to the rotating shaft and rotating therewith, and a microporous membrane filter fixedly connected to the telecentric end of the rotating arm ;
  • the microporous membrane filter comprises a liquid inlet, a liquid outlet, a membrane anterior chamber and a membrane posterior chamber respectively connected to the liquid inlet and the liquid outlet, and is disposed in the membrane anterior chamber and the chamber a membrane between the posterior chambers of the membrane;
  • the diameter of the filter pore of the filter membrane is smaller than the diameter of the cell to be separated;
  • the liquid inlet and the film front cavity are located at a telecentric end of the rotating arm, and the liquid outlet and the film back cavity are located at a proximal end of the rotating arm; so as to make water in the cell suspension And the biological particles and the biomolecules pass through the filter membrane due to the flow pressure, and the cells are blocked by the filter membrane and are separated from the filter membrane by the centrifugal force to settle in the membrane anterior chamber, and the cell-to-membrane is released. The blockage of the hole.
  • the arm has a length of 10-30 cm, a rotational speed of 500-1500 rpm, and a centrifugal force of 100 - 500 g.
  • the microporous membrane filter has a circular or square cross section.
  • the filter membrane has a diameter of l _30 um.
  • the filter membrane is made of a polyolefin or polyamide material.
  • the filter membrane is made of polyethylene, mixed cellulose, PE material or nylon material.
  • the liquid inlet of the microporous membrane filter is connected with an inlet pipe, and the inlet pipe is connected to an upstream pipe thereof through a rotary joint; the rotary joint is disposed on the axial center of the rotating arm On the upper fixed bracket,
  • a non-rotating end of the rotary joint is connected to an upstream pipe of the inlet pipe, and a rotating end of the rotary joint is connected to the microporous membrane filter through the liquid inlet pipe, so that the microporous membrane filter
  • the cell suspension filtration can be continuously performed while the shaft is rotated.
  • Another object of the present invention is to provide a cell separation system comprising the above-described centrifugal dynamic filter device structure, particularly a microporous membrane filter thereof, which can fully perform separation of target cells in a fully enclosed state.
  • the structure is simple, easy to operate, and closed throughout, which is not easy to cause pollution.
  • the disposable fully enclosed piping system comprises a microporous membrane filter, primary filtration , rotary joint, disposable syringe, balance liquid container, cell suspension container, enzyme liquid container connection pipe;
  • the microporous membrane filter includes a liquid inlet, a liquid outlet, a membrane front chamber and a membrane rear chamber respectively communicating with the liquid inlet and the liquid outlet, and is disposed in the membrane front chamber and the membrane a filter membrane between the rear chambers; a filter pore of the filter membrane is smaller than a size of the cells to be separated and extracted; the liquid inlet port and the membrane front cavity are located at a telecentric end of the centrifugal force of the microporous membrane filter.
  • the liquid outlet and the film back chamber are located at a proximal end of the centrifugal force of the microporous membrane filter, so that water and biological particles and biomolecules in the cell suspension pass through the filter due to liquid flow pressure.
  • the membrane is blocked by the filter membrane and is separated from the membrane by the centrifugal force to settle in the membrane anterior chamber, thereby clogging the membrane pores.
  • the connecting pipe includes a first pipe, a second pipe, a third pipe, a fourth pipe, a fifth pipe, and a sixth pipe;
  • the cell suspension container is inverted, and the bottle opening of the cell suspension container is connected to the first pipe; the primary filter is disposed in the first pipe;
  • the balance liquid container is inverted, the bottle mouth of the balance liquid container is connected to the second pipe, and the second pipe is connected to the first pipe;
  • One end of the third pipe is connected to the intersection of the first and second pipes, and the other end is connected to the disposable syringe;
  • One end of the fourth pipe is connected to the disposable syringe, and the other end is connected to the fixed end of the rotary joint;
  • One end of the fifth pipe is connected to the rotating end of the rotary joint, and the other end is connected to the liquid inlet of the microporous membrane filter;
  • One end of the sixth pipe communicates with the liquid outlet of the microporous membrane filter, and the other end opens to the waste liquid collection tank;
  • One end of the seventh pipe is connected to the intersection of the first and second pipes, and the other end is connected to the enzyme liquid container;
  • the instrument system comprises an electric rotating arm, an electric injection pump, and an equilibrium liquid heating and temperature control Device, cell suspension heating and temperature control device, cell suspension oscillation dial and electromagnetic control valve;
  • the arm end of the electric rotating arm fixes the microporous membrane filter, and the rotating shaft of the electric rotating arm is on the same straight line as the rotating shaft of the rotary joint;
  • the electric injection pump controls the disposable syringe;
  • the balance liquid heating and temperature control device is disposed outside the balance liquid container, and can warm and control the balance liquid therein;
  • the cell suspension is heated and controlled
  • the device is disposed outside the cell suspension container, and can warm and control the cell suspension therein;
  • the cell suspension container and the cell suspension heating and temperature control device are disposed on the cell suspension oscillation dial
  • the cell suspension oscillation dial can automatically oscillate the cell suspension container by a frequency set by a computer;
  • the electromagnetic control valve includes a first electromagnetic control valve, a second electromagnetic control valve, a third electromagnetic control valve, and a fourth electromagnetic control valve;
  • the first electromagnetic control valve is disposed in the first conduit and before the intersection of the first conduit and the second conduit;
  • the second electromagnetic control valve is disposed in the second conduit
  • the third electromagnetic control valve is disposed in the fourth conduit; and is between the disposable syringe and the rotary joint;
  • the fourth electromagnetic control valve is disposed in the seventh conduit.
  • the primary filter has a pore size larger than a target cell diameter, and the primary filter has a pore size of 200-300 mesh.
  • the electromagnetic control valves are electromagnetic pinch valves that control the opening and closing of the pipes.
  • the membrane front chamber and the membrane rear chamber of the microporous membrane filter are separated by the filter membrane, and the liquid inlet is disposed at a top or a side wall of the membrane front chamber.
  • the liquid port is disposed at the bottom or side wall of the back cavity of the film.
  • the filter membrane is a hydrophilic membrane.
  • the filter membrane has a diameter of l _30 um.
  • the filter membrane is made of a polyolefin or polyamide material.
  • the filter membrane is made of polyethylene, or mixed cellulose, or a PE material or a nylon material.
  • the centrifugal dynamic filtering device of the invention and the cell separation system using the microporous membrane filter thereof have the advantages of simple structure, convenient operation, rapid separation of cells, separation in a fully enclosed system, and difficulty in causing cells
  • the damage and contamination, separation of the whole process computer full automation control work is less demanding on the laboratory, and the advantages of other preferred features of the invention will be described in the following embodiments.
  • FIG. 1 is a perspective view of an embodiment of a centrifugal dynamic filtering device of the present invention.
  • FIG. 2 is a side elevational view of the centrifugal dynamic filter device of Figure 1.
  • Figure 3 is a side elevational view of the microporous membrane filter of the centrifugal dynamic filtration device of Figure 1.
  • Fig. 4 is a view showing the rotary joint of the inlet pipe for fixing the microporous membrane filter in the centrifugal dynamic filtering device shown in Fig. 1.
  • FIG 5 is a schematic illustration of a cell separation system including a microporous membrane filter in the centrifugal dynamic filtration device of Figure 1. detailed description
  • the centrifugal dynamic filtering device for cell separation of this embodiment includes a rotating shaft 28, which is vertically connected to the rotating shaft and rotates therewith.
  • the arm 211 is fixed to the microporous membrane filter 31 at the distal end of the arm.
  • the microporous membrane filter 31 includes a liquid inlet 311, a liquid outlet 312, a membrane front chamber 313 connected to the liquid inlet, a membrane rear chamber 314 connected to the liquid outlet, and a membrane front chamber 313 and a membrane.
  • the inlet 311 is disposed at the top end of the membrane front chamber 313, and the liquid outlet 312 is disposed at the bottom end of the membrane rear chamber 314, as shown in Fig. 3, so that the liquid can flow in the hydraulic pressure generated by the injection.
  • the filter membrane 315 is a hydrophilic filter membrane made of a polyolefin or polyamide material.
  • the filter membrane is made of polyethylene, or mixed cellulose, or a PE material or a nylon material.
  • the filter pores 3150 of the filter membrane 315 are smaller than the size of the cells to be separated, so that the cells to be extracted are left in the membrane anterior chamber 313, and the water and other biomolecules flow to the membrane posterior chamber 314, and then from the liquid outlet. 312 flows out. Since the size of the general somatic cells is 5-30 um, specifically, the diameter of the filter holes 3150 of the filter membrane 315 is less than 5 um. Preferably, the diameter of the filter pores of the filter membrane is from 1 to 30 ⁇ m; more preferably, the diameter of the filter pores 3150 is 3 ⁇ m to 5 ⁇ m.
  • the microporous membrane filter 31 is specifically a disc having a lumen and having a certain thickness; the liquid inlet surface of the membrane 315 is located at the telecentric end of the rotating arm, and the liquid discharging surface is located at the rotating arm. Near the heart. The rotation of the arm produces centrifugal force, so that the cells blocked by the filter membrane are "squeezed" away from the filter membrane due to the large centrifugal force of the particles, keeping the membrane pores 1350 unobstructed, making the water and useless or harmful biomolecules Flow through the membrane hole under the action of hydraulic pressure.
  • the membrane anterior chamber 3 13 is located at the telecentric end of the arm 2 1 1 and the membrane posterior chamber 3 14 is located at the proximal end of the arm 2 1 1 . That is, the position of the anterior membrane chamber 313 is farther from the position of the posterior chamber 314 than the posterior lumen 314.
  • the liquid flows from the liquid inlet 311 to the liquid outlet 312, and flows away from the rotating shaft 211 toward the proximal shaft 21 1; that is, the centrifugal force is opposite to the microporous membrane filter 31 when the rotating shaft 211 rotates.
  • the cells are separated from the membrane anterior chamber 313 by the membrane 315, and are separated from the membrane 315 by the centrifugal force, so that the membrane pores 3150 are not blocked, so that the filtration process Continue to be smooth.
  • the inlet pipe 375 is connected to its upstream pipe through a rotary joint 33.
  • the rotary joint 33 includes a non-rotating end 331 and a rotating end 332, and the non-rotating end 331 forms a cavity adapted to the rotating end 332, in which the rotating end 332 can rotate.
  • a seal ring 333 is provided at a portion where the non-rotating end 331 and the rotating end 332 are joined.
  • the non-rotating end 331 is fixed and communicates with the upstream pipe, and the connecting portion 3320 of the rotating end 332 communicates with the inlet pipe 375.
  • the inlet pipe 375 can be rotated following the rotation shaft 28 while being fixed at the non-rotating portion of the rotary joint. More preferably, the rotary joint 33 is disposed on the rotary shaft 28 or an extension thereof such that the inlet pipe 375 and the microporous membrane filter 31 are partially rotated in synchronization.
  • the system for extracting cells of the present embodiment includes a two-part one-time fully enclosed tubing system 3 and an instrument system 2.
  • the disposable fully enclosed piping system 3 comprises a microporous membrane filter 31, a primary filter 32, a rotary joint 33, a disposable syringe 34, a balance liquid container 35, a cell suspension container 36, an enzyme liquid container 38, a connecting pipe 37.
  • a microporous membrane filter 31 a primary filter 32, a rotary joint 33, a disposable syringe 34, a balance liquid container 35, a cell suspension container 36, an enzyme liquid container 38, a connecting pipe 37.
  • a primary filter 32 that filters out large particulate impurities in the cell suspension.
  • the primary filter 32 is disposed at the foremost end of the fluid direction, i.e., closest to the cell suspension holding device 36, such that during cleaning of the tissue and filtration, some relatively large impurities (such as Digested tissue fragments, macromolecules can be filtered out first.
  • the primary filter 32 is a 200 mesh filter, and in such a configuration, the filtering effect is relatively better.
  • 200-300 mesh filters are preferred.
  • any suitable configuration of filtration means can be used.
  • Rotating joint 33 the structure of which is as described above in the centrifugal dynamic filtering device, and is not described here.
  • Disposable syringe 34 driven by electric syringe pump 23 (described below), for pumping and injecting liquid.
  • the balance liquid container 35 which holds the balance liquid (also called buffer or washing liquid), and the balance liquid container 35 is maintained at 37 ° C by the heating and holding device 24.
  • the temperature of 37 °C is close to the body temperature, which is conducive to the protection of cells.
  • the temperature setting can also be adjusted by heating the holding device 24 according to the needs of cell protection.
  • the balance solution can be phosphate buffered saline (PBS) or sodium lactate.
  • the balance liquid is preferably selected from sodium lactate Grignard, because the electrolyte concentration, pH, osmotic pressure and the like are very close to the extracellular fluid, thereby facilitating cell survival and being suitable for washing residual collagen in the cell fluid. Enzymes that eliminate their harmful effects on cells.
  • a heat retaining device 25 is provided in the system of the present embodiment, which heats and maintains the cell suspension container 36 at a certain temperature. Generally, it is kept at a temperature of 37 °C. For uniform temperature, preferably, the heat retaining device 25 surrounds the outer wall of the container 36 surrounding the cell suspension.
  • the cell suspension holds the container 36, and the embodiment adopts an inverted cell holding container, the opening of which is Next.
  • the suspension holding container 36 is for accommodating raw materials for extracting cells obtained from a human body, and the raw materials may be various tissues including, but not limited to: adipose tissue, blood, bone marrow, muscle, skin, liver, sarcolemma, placenta. , umbilical cord, body fluid, secretion, cell culture fluid, etc.; in this embodiment, adipose tissue is separated by adipose tissue. These adipose tissue can be obtained by any suitable method in the prior art, such as liposuction (with a syringe) or lipectomy.
  • the amount of adipose tissue extracted depends on various factors, including: the ability of the extractor to provide fat and the amount of adipose stem cells required.
  • the cell suspension container 36 and the heat retention device 25 thereof are all located on the oscillating disk 26 having the oscillating device. The cell suspension 36 was shaken.
  • the enzyme solution container 38 is used for formulating and filling the liquid medicine required for the tissue treatment, and the present embodiment is a collagenase solution.
  • the connecting duct 37 includes a first duct 371, a second duct 371, a third duct 373, a fourth duct 374, a fifth duct 375, a sixth duct 376, and a seventh duct 377.
  • the first conduit 371 is connected to the mouth of the inverted cell suspension container 36; the first conduit 371 is provided with the primary filter 32.
  • the balance liquid container 35 is also inverted, and the bottle mouth of the balance liquid container 35 is connected to the second pipe 372, and the second pipe 372 is connected to the first pipe 371.
  • One end of the third duct 373 is connected to the intersection of the first and second ducts 371, 372, and the other end is connected to the disposable syringe 34.
  • One end of the fourth duct 374 is connected to the disposable syringe 34, and the other end is connected to the fixed end of the rotary joint 33.
  • One end of the fifth duct 375 communicates with the rotating end of the rotary joint 33, and the other end communicates with the liquid inlet of the microporous membrane filter 31.
  • One end of the sixth duct 376 is connected to the liquid outlet of the microporous membrane filter 31, and the other end opening extends to the waste liquid collection tank 27.
  • One end of the seventh pipe is connected to the intersection of the first and second pipes, and the other end is connected to the enzyme liquid container.
  • the pipes connecting the liquid inlet and the liquid outlet of the microporous membrane filter 31, that is, the fifth pipe 375 and the sixth pipe 376 can be directly cut and sealed by hot melt scissors.
  • the sealed microporous membrane filter 31 contains the required cells and can be directly stored and used.
  • each conduit may be rigid or flexible, depending on the particular needs.
  • each conduit is a flexible conduit, such as a polyethylene conduit typically used in clinical applications, or a silicone resin conduit, or a conduit for other materials known in the art.
  • the inner diameter of the pipe depends on the size and quantity of the liquid or tissue to be passed, and the required flow rate. These pipes must also be able to withstand the positive and negative pressures generated by the above-mentioned suction injection device.
  • the various components of the disposable fully enclosed tubing system 1 described above are single-use and fully enclosed, ensuring that the entire process of isolating cells from the cell suspension is carried out in a contaminated piping system.
  • the instrument system 2 of the cell separation system is described below, which is reusable.
  • the instrument system 2 includes an electric rotating arm 21, an electromagnetic control valve 22, an electric injection pump 23, an equilibrium liquid temperature control device 24, a cell suspension heating and temperature control device 25, and a cell suspension oscillation dial. 26.
  • the function arm end 211 of the electric rotating arm 21 fixes the above-mentioned microporous membrane filter 31, and the rotating shaft of the electric rotating arm 21 is on the same line as the rotating shaft of the rotary joint 33 of the system, so that the rotary joint 33 rotates in synchronization therewith.
  • the electric rotating arm 21 is provided as a balanced end 212, i.e., a counterweight is provided.
  • the electric rotating arm 21 is driven by a motor 28.
  • the electric syringe pump 23 controls the disposable syringe 34.
  • the balance liquid heating and temperature control device 24 is disposed outside the balance liquid container 35, and can warm and control the balance liquid therein.
  • the cell suspension heating and temperature control device 25 is disposed outside the cell suspension container 36, and can warm and control the cell suspension therein.
  • the cell suspension container 36 and the cell suspension heating and temperature control device 25 are disposed on the cell suspension oscillation disk 26 (not shown), and the cell suspension oscillation disk can automatically suspend the cells by the frequency set by the computer.
  • the liquid container 36 is oscillated.
  • the electromagnetic control valve 22 includes a first electromagnetic control valve 221, a second electromagnetic control valve 222, a third electromagnetic control valve 223, and a fourth electromagnetic control valve 224.
  • the first electromagnetic control valve 221 is disposed in the first duct 371 described above and before the intersection of the first duct 371 and the second duct 372.
  • the second electromagnetic control valve 222 is disposed in the second conduit 372.
  • the third solenoid control valve 223 is disposed in the fourth conduit 374; and is between the disposable syringe 34 and the rotary joint 33.
  • the fourth electromagnetic control valve 224 is disposed in the seventh duct 377.
  • the first, second, third, and fourth electromagnetic control valves 221, 222, 223, and 224 are electromagnetic pinch valves that can control the opening and closing of the pipe.
  • a disposable sealed piping system consisting of a microporous membrane filter 31, a primary filter 32, a rotary joint 33, a disposable syringe 34, a cell suspension container 36, a balance liquid container 35, an enzyme liquid container 38, and an associated conduit 37
  • the cell separation system can perform tissue cell washing, enzymatic digestion, cell filtration, separation, washing and collection.
  • the structure is simple, the required cells can be quickly obtained, the whole process can be closed, no pollution, and the adaptability is strong, which is favorable for the yield of cell extraction.
  • adipose tissue extracted from a human body is used as a raw material, and adipose stem cells need to be extracted therefrom.
  • the method includes the following steps (the valves not mentioned in the steps are all closed by default):
  • the balance liquid temperature control heating and holding device 24 is set at 37 ° C, and the bagged balance liquid 35 is placed in the temperature control heating and holding device 24 to preheat the balance liquid to 37 degrees, and the preheated 37 ° C balance liquid It can be used for the preparation of collagenase solution and provide suitable cell washing solution.
  • the disposable tubing system is placed in the instrument: the first conduit 371 is connected to the cell suspension container 36, the second conduit 372 is connected to the balance fluid container 35, the seventh conduit 377 is connected to the enzyme solution container 38, and the disposable syringe 34 is embedded in the electric device.
  • the syringe pump 23, the rotary joint 33 is placed on the holder, and the centrifugal filter 31 is placed on the boom function end 211.
  • the adipose tissue is loaded into the cell suspension container 36, which is a non-PVC infusion bag in this embodiment.
  • An equal volume of fat is extracted from the preheating balance liquid container 35, and injected into the enzyme solution container 38.
  • the enzyme diluent in the present embodiment is sodium lactate Ringer's solution, and an appropriate amount of collagenase is weighed according to the enzyme activity calibrated by the collagenase product specification. The two are mixed into a fat digestive enzyme solution, and the collagenase solution in the enzyme solution container 38 is injected into the cell suspension container 36.
  • the temperature-controlled warming and warming device 25 outside the container is set at a temperature of 37 °C.
  • the oscillating disc 26 carrying the container was operated at a rotational speed of 100 RPM for 20-40 minutes, and the preset enzymatic hydrolysis time was adjusted according to the enzyme activity and the degree of fat digestion.
  • the fat after being digested by the enzyme, is divided into three layers from bottom to top - an aqueous solution, a milk layer, and an oil layer.
  • Adipose stem cells are located in aqueous and milky layers. Since the bottom layer of the cell suspension device 36 is connected to the tube -371, the cell liquid filtration can be separated by a syringe pump.
  • the stratification may not be such a three layers, but the amount of the cell liquid is controllable, as long as the person skilled in the art can adjust accordingly. The purpose is to obtain cells.
  • the filtration, centrifugation, and separation processes begin.
  • the valve 221 is opened, and the water-soluble cell suspension of the lowermost layer of the cell suspension container 36 is extracted by the syringe 34 on the electric syringe pump 23.
  • the cell suspension passes through the primary filter 32, and the undigested tissues, impurities, and the like therein are filtered off.
  • A open the valve 222, and draw the appropriate amount of the 37 ° C balance liquid in the balance liquid holding device 35, which is 100 ml in this embodiment.
  • the valve 222 is closed, the valve 221 is opened, the balance solution is injected into the cell suspension container 36, and the process is repeated (2). Residual cells in the milky layer were obtained.
  • the valve 222 is opened, and the balance liquid is extracted from the treatment liquid accommodating device 35 by the syringe 34. Then, the valve 222 is closed, the valve 223 is opened, and the balance liquid flows into the dynamic centrifugal device through the pipe 374. Place the membrane anterior chamber of 31, and continuously dilute and rinse the cell fluid trapped in the anterior membrane of the membrane to completely remove harmful small molecules.
  • the washing liquid of this example was 150 ml.
  • the washing step is mainly to remove the residual enzyme in the cells or cell fluid.
  • the microporous membrane filter 31 is removed, and the solution and the solution therein can be used as it is.
  • the liquid inlet pipe and the liquid outlet pipe are directly melt-cut and sealed by a hot melt shear, and are stored for use. Before using directly, put the microporous membrane filter 31 into the oscillator and mix it to aspirate.
  • the cell separation method of this embodiment realizes a process of fully performing tissue digestion, preliminary filtration, dynamic filtration to obtain cells, and collection of cells. Moreover, the whole process is automatically completed in a one-time sealed pipeline, without being exposed to the external environment, avoiding pollution, reducing mechanical damage of cells during operation, and making the survival rate of the separated cells high.
  • the design of the piping system may be different from that in the above embodiment as long as the corresponding functions can be realized.

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Abstract

本发明公开了一种用于活细胞分离的离心动态过滤装置，包括转轴（28）、垂直连接于转轴（28）且随其旋转的转臂（211）、固定连接于转臂（211）远心端的微孔膜过滤器（31）。微孔膜过滤器（31）包括进液口（311）、出液口（312），分别连通于进液口（311）和出液口（312）的膜前腔（313）和膜后腔（314），以及设置于两腔之间的滤膜（315）；滤膜（315）的滤孔小于要分离的细胞的大小。本发明还公开了细胞分离系统。

Description

离心动态过滤装置及利用其的细胞分离系统
技术领域
本发明涉及生物细胞分离领域， 尤其涉及一种离心动态过滤装置及包括其 微孔膜过滤器的细胞分离系统， 利用该系统可建立细胞快速分离方法。
背景技术
治疗疾病的最好方法之一就是利用活组织重建和更新疾病及老化的组织； 这种治疗方法被称为 "细胞治疗"。 细胞治疗已有百年历史， 目前广泛地应用于 肿瘤、 肝脏疾病、 去疤痕等各个领域， 并且具有广阔的发展前景。
细胞治疗最基础的一个问题就是先要分离出目标细胞。 在现有技术中， 细 胞的分离均采用离心机来实现； 这种分离方法不仅操作繁琐； 而且由于操作时 需要多次反复取液加液， 容易造成细胞机械性损伤且易污染， 对实验室的环境 要求非常严格， 从而影响细胞质量， 增加细胞分离成本。
为此， 需要提供一种更好的用于细胞分离的装置、 系统和方法。
发明内容
本发明的目的之一在于提供一种离心动态过滤装置， 其结构简单， 操作方 便， 能快速分离细胞， 且分离能在全封闭系统内进行， 减轻分离过程产生的细 胞损伤， 不易造成细胞污染， 分离全过程可以计算机全自动化控制。
为达到上述目的， 本发明采用如下技术方案：
一种离心动态过滤装置， 用于活细胞分离， 其特征在于： 包括转 轴、 垂直连接于所述转轴且随其旋转的转臂、 固定连接于所述转臂的 远心端的微孔膜过滤器；
其中， 所述微孔膜过滤器包括进液口、 出液口、 分别连通于所述 进液口和所述出液口的膜前腔和膜后腔、 设置于所述膜前腔和所述膜 后腔之间的滤膜； 所述滤膜的滤孔的直径小于要分离细胞的直径；
所述进液口及所述膜前腔位于所述转臂的远心端，所述出液口及 所述膜后腔位于所述转臂的近心端； 以使细胞悬浮液中水份和生物颗 粒及生物分子由于液流压通过所述滤膜， 而细胞被所述滤膜阻挡并在 离心力的作用下甩离所述滤膜而沉降于所述膜前腔， 解除了细胞对膜 孔的堵塞。
优选地， 所述转臂长度 10-30cm， 转速 500- 1500转 /分， 产生的 离心力为 100 - 500g。
优选地， 所述微孔膜过滤器的横截面为圆形或方形。
优选地， 所述滤膜的滤孔的直径为 l _30um。
优选地， 所述滤膜采用聚烯烃类或聚酰胺类材料制成。
优选地， 所述滤膜由聚乙烯、 混合纤维素、 PE材料或尼龙材料制 成。
优选地， 所述微孔膜过滤器的所述进液口连接有进液管， 所述进 液管通过旋转接头连接于其上游管道； 所述旋转接头设置于所述转臂 的轴心正上方的固定支架上，
所述旋转接头的非旋转端连接连通所述进液管的上游管道， 所述 旋转接头的旋转端通过所述进液管连接于所述微孔膜过滤器， 使得所 述微孔膜过滤器能在所述转轴转动时连续地进行细胞悬浮液过滤。
本发明的目的之二在于提供一种包括上述离心动态过滤装置结 构尤其其微孔膜过滤器的细胞分离系统， 其能在全封闭状态下全自动 完成目标细胞的分离。 其结构简单， 便于操作， 并且全程封闭， 不易 造成污染。
为达到上述目的， 本发明采用如下技术方案：
一种细胞分离系统， 其特征在于包括：
一次性全封闭管道系统和仪器系统；
其中， 所述一次性全封闭管道系统包括微孔膜过滤器、 初级过滤 器、 旋转接头、 一次性注射器、 平衡液容器、 细胞悬浮液容器、 酶液 容器连接管道；
所述微孔膜过滤器包括进液口、 出液口、 分别连通于所述进液口 和所述出液口的膜前腔和膜后腔、 设置于所述膜前腔和所述膜后腔之 间的滤膜； 所述滤膜的滤孔小于要分离提取的细胞的大小； 所述进液 口及所述膜前腔位于所述微孔膜过滤器所受离心力的远心端，所述出 液口及所述膜后腔位于所述微孔膜过滤器所受离心力的近心端， 以使 细胞悬浮液中水份和生物颗粒及生物分子由于液流压通过所述滤膜， 而细胞被所述滤膜阻挡并在离心力的作用下甩离所述滤膜而沉降于所 述膜前腔， 解除了细胞对膜孔的堵塞。
所述连接管道包括第一管道、 第二管道、 第三管道、 第四管道、 第五管道、 第六管道；
所述细胞悬浮液容器为倒置的， 所述细胞悬浮液容器的瓶口处连 接连通所述第一管道； 所述第一管道中设置所述初级过滤器；
所述平衡液容器为倒置的， 所述平衡液容器的瓶口处连接连通所 述第二管道， 所述第二管道连通所述第一管道；
所述第三管道的一端连通于所述第一、 二管道交汇处， 且另一端 连通所述一次性注射器；
所述第四管道的一端连通于所述一次性注射器， 且另一端连通于 所述旋转接头的固定端；
所述第五管道的一端连通于所述旋转接头的旋转端， 且另一端连 通于所述微孔膜过滤器的进液口；
所述第六管道的一端连通于所述微孔膜过滤器的出液口， 且另一 端开口伸至废液收集槽；
所述第七管道一端连通于所述第一、 二管道交汇处， 且另一端连 通于所述酶液容器；
其中， 所述仪器系统包括电动旋转臂、 电动注射泵、 平衡液加温控温 装置、 细胞悬浮液加温控温装置、 细胞悬浮液振荡转盘以及电磁控制 阀；
所述电动旋转臂的臂端固定所述微孔膜过滤器， 所述电动旋转臂 的旋转轴与所述旋转接头的转轴在同一条直线上；
所述电动注射泵控制所述一次性注射器； 所述平衡液加温控温装 置设置在所述平衡液容器外， 能对其中的平衡液加温控温； 所述细胞 悬浮液加温控温装置设置于所述细胞悬浮液容器外， 能对其中的细胞 悬浮液加温控温； 所述细胞悬浮液容器及所述细胞悬浮液加温控温装 置设置于所述细胞悬浮液振荡转盘上， 所述细胞悬浮液振荡转盘能通 过计算机设置的频率自动地对所述细胞悬浮液容器进行振荡；
所述电磁控制阀包括第一电磁控制阀、 第二电磁控制阀、 第三电 磁控制阀、 第四电磁控制阀；
所述第一电磁控制阀设置在所述第一管道中， 且处于所述第一管 道和所述第二管道的交汇处之前；
所述第二电磁控制阀设置在所述第二管道中；
所述第三电磁控制阀设置在所述第四管道中； 且其处于所述一次 性注射器与旋转接头之间；
所述第四电磁控制阀设置在所述第七管道中。
优选地， 所述初级过滤器的滤孔孔径大于目标细胞直径， 所述初 级过滤器微孔膜孔径为 200-300 目。 优选地， 电磁控制阀均为电磁夹 管阀， 能控制管道的开放与关闭。
优选地， 所述微孔膜过滤器的所述膜前腔和所述膜后腔由所述滤 膜分隔而成， 所述进液口设置在所述膜前腔的顶部或侧壁， 所述出液 口设置在所述膜后腔的底部或侧壁。
优选地， 所述滤膜为亲水性膜。
优选地， 所述滤膜的滤孔的直径为 l _30um。
优选地， 所述滤膜采用聚烯烃类或聚酰胺类材料制成。 优选地， 所述滤膜由聚乙烯、 或混合纤维素、 或 PE材料或尼龙材料制成。
本发明的有益效果在于： 本发明的离心动态过滤装置及利用其微孔膜过滤 器的细胞分离系统， 其结构简单， 操作方便， 能快速分离细胞， 分离在全封闭 系统内进行， 不易造成细胞的损伤和污染， 分离全过程计算机全自动化控制工 作对实验室的要求较低， 本发明的其它优选特点的优越性将在下文实施方式中 描述。
附图说明
图 1为本发明离心动态过滤装置的一个实施例的立体示意图。
图 2为图 1所示离心动态过滤装置的侧面示意图。
图 3为图 1所示离心动态过滤装置中的微孔膜过滤器的侧面示意图。
图 4为图 1所示离心动态过滤装置中固定微孔膜过滤器的进液管的旋转接 头的示意图。
图 5为包括图 1所示离心动态过滤装置中的微孔膜过滤器的细胞分离系统 的示意图。 具体实施方式
下面结合附图， 详细介绍本发明的优选实施方式。 下述优选实施例仅仅起 到示例的作用而已，并不能作为对本发明保护范围的限制。
(一） 离心动态过滤装置
如图 1、 图 2、 图 3所示为本发明一个实施例的离心动态过滤装置， 该实施 例的用于细胞分离的离心动态过滤装置包括转轴 28、 垂直连接于转轴且随其旋 转的转臂 211、 固定连接于转臂的远心端的微孔膜过滤器 31。
其中， 微孔膜过滤器 31包括进液口 311、 出液口 312、 连通于进液口的膜 前腔 313、 连通于出液口的膜后腔 314、 设置于膜前腔 313和膜后 314之间的滤 膜 315。优选地， 进液 311设置在膜前腔 313的顶端， 出液口 312设置在膜后腔 314的底端， 如图 3所示， 这样， 液体能在注射产生的液压流动。 其中， 滤膜 315 为亲水性过滤膜， 其采用聚烯烃类或聚酰胺类材料制成。 优选地， 滤膜由聚乙烯、 或混合纤维素、 或 PE材料或尼龙材料制成。 滤膜 315 的滤孔 3150小于要分离提取的细胞的大小， 从而使得要提取的细胞被隔离留存 在膜前腔 313，而水份及其它生物分子流到膜后腔 314，然后从出液口 312流出。 由于一般体细胞的大小为 5-30um， 具体地， 滤膜 315的滤孔 3150的直径小于 5um。 优选地， 滤膜的滤孔的直径为 l-30um; 更优选地， 滤孔 3150 的直径为 3um_5um。
优选地， 在本实施例中， 微孔膜过滤器 31具体为具有内腔的且有一定厚度 的圆饼状； 滤膜 315 的进液面位于转臂远心端， 出液面位于转臂近心端。 转臂 转动产生离心力， 从而使得被滤膜阻挡的细胞因其颗粒大受到更大的离心力而 被 "甩"离滤膜， 保持了膜孔 1350通畅， 使水份及无用或有害的生物分子在液 压作用下通过膜孔流走。
优选地， 膜前腔 3 13位于转臂 2 1 1 的远心端， 膜后腔 3 14位于转臂 2 1 1 的近心端。 也就是说， 膜前腔 313的位置与膜后腔 314的位置相比， 膜 前腔 313离上述转轴 211比膜后腔 314远。 这样， 液体从进液口 311流向出液 口 312， 是从远离转轴 211处向近转轴 21 1方向流动； 亦即， 与该微孔膜过滤器 31在转轴 211旋转时所受到的离心力方向相反。 从而使得在细胞悬液到达膜前 腔 313时， 细胞被滤膜 315隔离在膜前腔 313的同时， 会在离心力的作用下远 离滤膜 315， 从而不会堵住滤孔 3150， 使得过滤过程持续顺畅。
在本实施例中， 进液管 375通过旋转接头 33连接于其上游管道。 其中具体 地， 如图 4所示， 旋转接头 33包括非旋转端 331和旋转端 332， 非旋转端 331 形成与旋转端 332适配的内腔， 旋转端 332能在该内腔内旋转。在非旋转端 331 和旋转端 332接合的部位设有密封圈 333。非旋转端 331被固定且连通于上游管 道， 其旋转端 332的连接部位 3320连通于进液管 375。 这样， 进液管 375在旋 转接头的非旋转部位固定的同时， 能跟随转轴 28—起转动。 更优选地， 旋转接 头 33设置在转轴 28上或其延长线上， 使得进液管 375和微孔膜过滤器 31部分 同步旋转。
(二） 细胞分离系统 下面将介绍本发明细胞分离系统的优选实施例。 如图 5所示， 本实施例的提取细胞的系统， 包括两大部分一一次性全封 闭管道系统 3和仪器系统 2。
其中， 一次性全封闭管道系统 3 包括微孔膜过滤器 3 1、 初级过滤 器 32、 旋转接头 33、 一次性注射器 34、 平衡液容器 35、 细胞悬浮液 容器 36、 酶液容器 38、 连接管道 37。 下面分别来介绍。
( 1 )微孔膜过滤器 31，其结构如上文离心动态过滤装置所述，在此不累述。
( 2 )初级过滤器 32， 其能滤掉细胞悬液中大颗粒杂质。 在本实施例中， 该 初级过滤器 32设置在流体方向的最前端， 即最靠近细胞悬液盛放装置 36， 从而 使得在清洗组织和过滤的过程中， 一些相对较大的杂质 （比如未消化的组织片 断、 大分子） 能被首先过滤出去。 优选地， 在本实施例中， 优选地， 经过多次 试验， 初级过滤器 32为 200目过滤器， 这样的配置下， 过滤效果相对更好。 而 且 200-300 目过滤器都是较佳的。 当然， 在本发明其他的实施例中， 任何合适 结构的过滤装置也可以使用。
( 3 ) 旋转接头 33， 其结构如上文离心动态过滤装置中所述， 在此不累述。
(4 ) 一次性注射器 34， 在电动注射泵 23 (如下文所述） 的驱动下， 用于 抽取和注射液体。
( 5 ) 平衡液容器 35， 盛放平衡液 （也叫缓冲液或洗涤液）， 平衡液容器 35 被加热保温装置 24保持在 37°C。 37°C的温度接近人体体温， 有利于对细胞的保 护。 也可根据细胞保护需要， 通过加热保温装置 24调整温度设置。
这里的平衡液， 可以采用磷酸盐缓冲液 （PBS ) , 也可以用乳酸钠格林氏液。 在本实施例中， 平衡液优选地选用乳酸钠格林氏液， 原因在于其电解质浓度、 PH、 渗透压等与细胞外液非常接近，因而更有利于细胞存活， 适用于清洗细胞液 中残留的胶原酶， 消除其对细胞的有害影响。 优选地， 由于在提取细胞的过程 中需要将原料保持在接近体温的温度， 因此在本实施例的系统中设置有保温装 置 25， 其将细胞悬液盛放容器 36 加热并保持在一定的温度， 一般来讲保持在 37°C的温度。 为了温度的均匀， 优选地， 该保温装置 25环绕包围着细胞悬液盛 放容器 36的外壁。
( 6 )细胞悬液盛放容器 36， 本实施例采用倒置的细胞盛放容器， 其开口向 下。 该悬浮液盛放容器 36用于容纳从人体获得的用于提取细胞的原料， 原料可 以是各种组织， 包括但不限于： 脂肪组织、 血液、 骨髓、 肌肉、 皮肤、 肝脏、 肌膜、 胎盘、 脐带、 体液、 分泌物、 细胞培养液等； 在本实施例中便是采用脂 肪组织分离脂肪干细胞。 这些脂肪组织可以采用现有技术中任何合适的方法来 获得， 比如可以采用抽脂术 （用注射器） 或者脂肪切除术。 脂肪组织的抽取数 量取决于各种因素， 包括： 被抽取者提供脂肪的能力和需求脂肪干细胞数量。 优选地， 为了加快胶原酶液与脂肪组织的混合、 加快酶对脂肪组织的消化， 在 本实施例的系统中细胞悬液容器 36及其保温装置 25均位于有振荡装置的振荡 盘 26上， 对细胞悬浮液 36进行振荡。
( 7 )酶液容器 38， 用于配制和加注组织处理过程中所需药液， 本实施例为 胶原酶液。
( 8 ) 连接管道 37包括第一管道 371、 第二管道 371、 第三管道 373、 第四 管道 374、 第五管道 375、 第六管道 376、 第七管道 377。
其中， 第一管道 371连接连通上述倒置的细胞悬浮液容器 36的瓶口处； 第 一管道中 371设置上述初级过滤器 32。
上述平衡液容器 35也为倒置的， 平衡液容器 35的瓶口处连接连通第二管 道 372， 第二管道 372连通第一管道 371。
第三管道 373的一端连通于第一、 二管道 371、 372的交汇处， 且另一端 连通一次性注射器 34。
第四管道 374 的一端连通于一次性注射器 34， 且另一端连通于旋转接头 33的固定端。
第五管道 375的一端连通于旋转接头 33的旋转端， 且另一端连通于微孔 膜过滤器 31的进液口。 第六管道 376的一端连通于微孔膜过滤器 31的出液口， 且另一端开口伸至 废液收集槽 27。
第七管道一端连通于第一、 二管道交汇处， 且另一端连通于酶液容器。 在本系统中， 分离细胞完毕之后， 可以直接用热熔剪刀将微孔膜过滤器 31 的进液口和出液口连接的管道 - 即第五管道 375和第六管道 376剪断并封口， 该密封的微孔膜过滤器 31中存留有需要的细胞， 能直接存放使用。
上述各个管道可以是硬质的， 也可以是柔性的， 视具体需要而定。 在本优 选实施例中， 各个管道为柔性管道， 比如临床应用中通常使用的聚乙烯管道， 或者硅有机树脂管道， 或者现有技术中已知的其他材料的管道。 管道的内径大 小取决于要通过的液体或者组织的大小、 数量， 以及需要的流速等。 这些管道 还必须能承受因上述抽吸注射装置所产生的正负压。
上述一次性全封闭管道系统 1 的各个组成部分都是一次性使用的， 而且全封闭， 保证从细胞悬浮液中分离出细胞的全过程不受污染的管 道系统中进行。
下面介绍细胞分离系统的仪器系统 2， 它是可重复利用的。
如图 5所示， 该仪器系统 2包括电动旋转臂 21、 电磁控制阀 22、 电动注射 泵 23、 平衡液加温控温装置 24、 细胞悬浮液加温控温装置 25、 细胞悬浮液振荡 转盘 26。
电动旋转臂 21的功能臂端 211固定上述微孔膜过滤器 31， 电动旋转臂 21 的旋转轴与该系统的旋转接头 33的转轴在同一条直线上， 以让旋转接头 33随 其同步旋转。 在功能臂端的相反端， 该电动旋转臂 21设置为平衡端 212， 即设 有平衡块。 电动旋转臂 21由电机 28驱动。
电动注射泵 23控制一次性注射器 34。
平衡液加温控温装置 24设置在平衡液容器 35外， 能对其中的平衡液加温 控温。
细胞悬浮液加温控温装置 25设置于细胞悬浮液容器 36外， 能对其中的细 胞悬浮液加温控温。 此外， 细胞悬浮液容器 36及细胞悬浮液加温控温装置 25 设置于细胞悬浮液振荡盘 26 (图中未示出） 上， 细胞悬浮液振荡转盘能通过计 算机设置的频率自动地对细胞悬浮液容器 36进行震荡。
电磁控制阀 22包括第一电磁控制阀 221、 第二电磁控制阀 222、 第三电磁 控制阀 223、 第四电磁控制阀 224。
第一电磁控制阀 221设置在上述第一管道 371中， 且处于第一管道 371和 第二管道 372的交汇处之前。
第二电磁控制阀 222设置在第二管道 372中。 第三电磁控制阀 223设置在第四管道 374中； 且其处于一次性注射器 34与 旋转接头 33之间。
第四电磁控制阀 224设置在第七管道 377中。
上述第一、 第二、 第三、 第四电磁控制阀 221、 222、 223、 224均为电磁夹 管阀， 能控制管道的开放与关闭。
由微孔膜过滤器 31、 初级过滤器 32、 旋转接头 33、 一次性注射器 34、 细 胞悬浮液容器 36、 平衡液容器 35、 酶液容器 38及相连管道 37组成的一次性密 封管道系统与由电动离心转臂 21、 电动注射泵 23、 电磁夹管阀 22、 平衡液加温 控温装置 24、 细胞悬浮液加温控温装置 25细胞悬浮液振荡转盘 26构成的仪器 系统， 共同形成的上述细胞分离系统， 能完成组织细胞的清洗、 酶解消化、 细 胞的过滤分离洗涤及收集等工作。 结构简单， 能快速获得所需细胞， 可全程封 闭， 无污染， 适应性强， 有利于细胞提取的产量化。
(三） 细胞分离方法
下面对应上述第一个实施例的细胞分离系统， 介绍利用该系统的细胞分离 方法。 通过该方法的阐释， 上述系统的结构和功能也能得到更好的理解。
本实施例的细胞分离方法， 以从人体抽取的脂肪组织为原料， 需要从中提 取脂肪干细胞。 该方法包括如下步骤 （步骤中未提到的阀门均默认为关闭的）：
1、 预热平衡液， 连接一次性容器、 过滤器、 注射器和管道系统
将平衡液温控加温保温装置 24设定 37°C， 把袋装平衡液体 35放置在温控 加温保温装置 24中， 使平衡液预热到 37度， 预热的 37 °C平衡液既能备用于胶 原酶液配制， 又能提供合适的细胞洗涤液。 把一次性管道系统置于仪器： 第一 管道 371接入细胞悬液容器 36，第二管道 372接入平衡液容器 35，第七管道 377 接于酶液容器 38， 把一次性注射器 34嵌入电动注射泵 23， 旋转接头 33置于固 定架， 离心过滤器 31置于转臂功能端 211。
2、 脂肪组织酶解
将脂肪组织装入细胞悬液容器 36， 本实施例该容器为非 PVC输液袋。 从预 热平衡液容器 35抽取于脂肪等体积的平衡液， 注入酶液容器 38， 本实施例酶稀 释液为乳酸钠林格氏液， 根据胶原酶产品说明书标定的酶活性称取适量胶原酶， 两者混合为脂肪消化酶液， 将酶液容器 38内的胶原酶液注入细胞悬液容器 36。 该容器外的温控加温保温装置 25设置温度为 37°C。 承载该容器的振荡盘 26工 作， 转速为 100RPM, 时间 20-40分钟， 根据酶活性及脂肪消化程度调整预设酶 解时间。
脂肪， 被酶消化后， 分成自下而上的三层 -水溶液、 乳状层、 油层。 脂肪 干细胞位于水溶液及乳状层内。 由于细胞悬液装置 36最底层与管道 -371连接， 通过注射泵定量设置， 就可进行细胞液内细胞过滤分离。 当然， 在本发明其他 的实施例中， 原料为其他组织或者组织片断等时， 分层可能就不是这样的三层， 但细胞液量是可控的， 只要本领域技术人员相应地调整即可， 目的就是要获得 含细胞。
3、利用过滤装置过滤杂质和大分子， 利用微孔膜过滤器动态过滤分离提取 细胞
(1)按照计算机预设参数， 过滤、 离心、 分离过程开始工作。 开启阀门 221， 利用电动注射泵 23上的注射器 34， 抽取细胞悬浮液容器 36最下层的水溶性细 胞悬浮液。 此时， 细胞悬浮液通过初级过滤器 32， 将其中的未消化的组织、 杂 质等过滤掉。
(2)关闭阀门 221， 打开阀门 223， 利用电动注射泵上的注射器 34将其内的 溶液通过管道四 374、 旋转接头 33、 管道五 375注入转动中的离心动态过滤装 置 31， 到达膜前腔， 水份及小的生物分子， 特别是胶原酶经膜孔流入膜后腔， 经管道六 376， 排入废液槽 27， 细胞截留在膜前腔， 且由于离心力差别， 细胞 甩离膜孔， 不会形成膜孔堵塞。 本实施例转臂半径为 20， 转速为 1500转 /分。
(3)重复抽吸过程，根据计算机对量的预设，直到油水分离面以下溶液抽完。 为提高细胞分离效率； 可以重复注入平衡液并再次过滤， 具体步骤包括：
A，开启阀门 222， 吸取平衡液容纳装置 35中的 37°C 平衡液适量， 本实施 例为 100ml。 关闭阀门 222， 打开阀门 221， 将平衡液注入细胞悬液容器 36， 重 复 （2 ) 过程。 获得乳状层内残留细胞。
4、 平衡液洗涤离心动态过滤装置中提取到的细胞
首先， 开启阀门 222， 利用注射器 34从处理液容纳装置 35中抽取平衡液。 然后， 关闭阀门 222， 开启阀门 223、 平衡液经管道四 374流入动态离心装 置 31的膜前腔， 连续稀释冲洗截留于膜前腔的细胞液， 彻底去除有害的小分子 物质。 本实施例洗涤液为 150ml。
洗涤步骤主要是为了去除细胞或细胞液中残存的酶。
5、 取下微孔膜过滤器 31， 封闭管道待用。
将微孔膜过滤器 31取下， 其内的及溶液就可以直接使用。 在本实施例中， 采用热熔剪直接将进液管道和出液管道热熔剪断并封口， 保存待用。 直接使用 前将微孔膜过滤器 31放入振荡器振荡混匀即可吸出。
该实施例的细胞分离方法实现了全自动完成组织的消化、 初步过滤、 动态 过滤获取细胞以及细胞的收集等工序。 而且全程均一次性密封管道中自动完成， 不用暴露在外界环境下， 避免了污染， 减轻了操作中细胞的机械损伤， 使分离 细胞的存活率高。
当然， 在本发明的其他实施例中， 管道系统的设计可以与上述实施例中的 不同， 只要能实现相应的功能即可。
以上描述的是本发明的优选实施例而已， 不能以此限制本发明的保护范围。 任何对本发明技术方案的等同变换和替换， 都在本发明的保护范围之内。

Claims

权 利 要 求 书
1、 一种离心动态过滤装置， 用于活细胞分离， 其特征在于： 包 括转轴、 垂直连接于所述转轴且随其旋转的转臂、 固定连接于所述转 臂远心端的微孔膜过滤器；
其中， 所述微孔膜过滤器包括进液口、 出液口、 分别连通于所述 进液口和所述出液口的膜前腔和膜后腔、 设置于所述膜前腔和所述膜 后腔之间的滤膜；
所述滤膜的滤孔的直径小于要分离细胞的直径；
所述进液口及所述膜前腔位于所述转臂的远心端，所述出液口及 所述膜后腔位于所述转臂的近心端； 以使细胞悬浮液中水份和生物颗 粒及生物分子由于液流压通过所述滤膜， 而细胞被所述滤膜阻挡并在 离心力的作用甩离所述滤膜而沉降于所述膜前腔。
2、 如权利要求 1 所述的离心动态过滤装置， 其特征在于： 所述 转臂长度 10-30cm， 转速 500- 1500转 /分， 产生的离心力为 100_500g。
3、 如权利要求 1 所述的离心动态过滤装置， 其特征在于： 所述 微孔膜过滤器的横截面为圆形或方形。
4、 如权利要求 1 所述的离心动态过滤装置， 其特征在于： 所述 滤膜的滤孔的直径为 l _30um。
5、 如权利要求 1 所述的离心动态过滤装置， 其特征在于： 所述 滤膜采用聚烯烃类或聚酰胺类材料制成。
6、 如权利要求 5 所述的离心动态过滤装置， 其特征在于： 所述 滤膜由聚乙烯、 混合纤维素、 PE材料或尼龙材料制成。
7、 如权利要求 1 所述的离心动态过滤装置， 其特征在于： 所述 微孔膜过滤器的所述进液口连接有进液管， 所述进液管通过旋转接头 连接于其上游管道； 所述旋转接头设置于所述转臂的轴心正上方的固 定支架上，
所述旋转接头的非旋转端连接连通所述进液管的上游管道， 所述 旋转接头的旋转端通过所述进液管连接于所述微孔膜过滤器， 使得所 述微孔膜过滤器能在所述转轴转动时连续地进行细胞悬浮液过滤。
8、 一种细胞分离系统， 其特征在于包括：
一次性全封闭管道系统和仪器系统；
其中， 所述一次性全封闭管道系统包括微孔膜过滤器、 初级过滤 器、 旋转接头、 一次性注射器、 平衡液容器、 细胞悬浮液容器、 酶液 容器连接管道；
所述微孔膜过滤器包括进液口、 出液口、 分别连通于所述进液口 和所述出液口的膜前腔和膜后腔、 设置于所述膜前腔和所述膜后腔之 间的滤膜； 所述滤膜的滤孔小于要分离提取的细胞的大小； 所述进液 口及所述膜前腔位于所述微孔膜过滤器所受离心力的远心端，所述出 液口及所述膜后腔位于所述微孔膜过滤器所受离心力的近心端， 以使 细胞悬浮液中水份和生物颗粒及生物分子由于液流压通过所述滤膜， 而细胞被所述滤膜阻挡并在离心力的作用下甩开所述滤膜而沉降于所 述膜前腔；
所述连接管道包括第一管道、 第二管道、 第三管道、 第四管道、 第五管道、 第六管道、 第七管道；
所述细胞悬浮液容器为倒置的， 所述细胞悬浮液容器的瓶口处连 接连通所述第一管道； 所述第一管道中设置所述初级过滤器；
所述平衡液容器为倒置的， 所述平衡液容器的瓶口处连通所述第 二管道， 所述第二管道连通所述第一管道；
所述第三管道的一端连通于所述第一、 二管道交汇处， 且另一端 连通所述一次性注射器；
所述第四管道的一端连通于所述一次性注射器， 且另一端连通于 所述旋转接头的固定端；
所述第五管道的一端连通于所述旋转接头的旋转端， 且另一端连 通于所述微孔膜过滤器的进液口；
所述第六管道的一端连通于所述微孔膜过滤器的出液口， 且另一 端开口伸至废液收集槽；
所述第七管道一端连通于所述第一、 二管道交汇处， 且另一端连 通于所述酶液容器；
其中， 所述仪器系统包括电动旋转臂、 电动注射泵、 平衡液加温 控温装置、 细胞悬浮液加温控温装置、 细胞悬浮液振荡转盘以及电磁 控制阀；
所述电动旋转臂的臂端固定所述微孔膜过滤器， 所述电动旋转臂 的旋转轴与所述旋转接头的转轴在同一条直线上；
所述电动注射泵控制所述一次性注射器； 所述平衡液加温控温装 置设置在所述平衡液容器外， 能对其中的平衡液加温控温； 所述细胞 悬浮液加温控温装置设置于所述细胞悬浮液容器外， 能对其中的细胞 悬浮液加温控温； 所述细胞悬浮液容器及所述细胞悬浮液加温控温装 置设置于所述细胞悬浮液振荡转盘上， 所述细胞悬浮液振荡转盘能通 过计算机设置的频率自动地对所述细胞悬浮液容器进行振荡；
所述电磁控制阀包括第一电磁控制阀、 第二电磁控制阀、 第三电 磁控制阀、 第四电磁控制阀；
所述第一电磁控制阀设置在所述第一管道中， 且处于所述第一管 道和所述第二管道的交汇处之前；
所述第二电磁控制阀设置在所述第二管道中； 所述第三电磁控制阀设置在所述第四管道中； 且其处于所述一次 性注射器与旋转接头之间；
所述第四电磁控制阀设置于所述第七管道。
9、 如权利要求 8所述的细胞分离系统， 其特征在于： 所述初级 过滤器的滤孔孔径大于目标细胞直径， 所述初级过滤器微孔膜孔径为 200-300 目。
10、 如权利要求 8所述的细胞分离系统， 其特征在于： 电磁控制 阀均为电磁夹管阀， 能控制管道的开放与关闭。
1 1、 如权利要求 8所述的细胞分离系统， 其特征在于： 所述微孔 膜过滤器的所述膜前腔和所述膜后腔由所述滤膜分隔而成， 所述进液 口设置在所述膜前腔的顶部或侧壁， 所述出液口设置在所述膜后腔的 底部或侧壁。
12、 如权利要求 8所述的细胞分离系统， 其特征在于： 所述滤膜 为亲水性膜。
13、 如权利要求 8所述的细胞分离系统， 其特征在于： 所述滤膜 的滤孔的直径为 l _30um。
14、 如权利要求 10 所述的细胞分离系统， 其特征在于： 所述滤 膜采用聚烯烃类或聚酰胺类材料制成。
15、 如权利要求 14 所述的细胞分离系统， 其特征在于： 所述滤 膜由聚乙烯、 或混合纤维素、 或 PE材料或尼龙材料制成。
PCT/CN2012/086694 2012-12-14 2012-12-14 离心动态过滤装置及利用其的细胞分离系统 WO2014089838A1 (zh)

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CA2873077A CA2873077C (en) 2012-12-14 2012-12-14 Centrifugal filtration device and cell separation system and methods of use thereof
AU2012396668A AU2012396668B2 (en) 2012-12-14 2012-12-14 Centrifugal dynamic filtering apparatus and cell separation system using same
MYPI2014003212A MY171082A (en) 2012-12-14 2012-12-14 Centrifugal filtration device and cell separation system with the same field of the invention
SG11201408169YA SG11201408169YA (en) 2012-12-14 2012-12-14 Centrifugal dynamic filtering apparatus and cell separation system using same
RU2014144222/10A RU2600871C2 (ru) 2012-12-14 2012-12-14 Устройство для центробежного фильтрования и система отделения клеток с таким устройством для центробежного фильтрования
CN201280063057.5A CN104363972B (zh) 2012-12-14 2012-12-14 离心动态过滤装置及利用其的细胞分离系统
EP12890047.9A EP2933326B1 (en) 2012-12-14 2012-12-14 Centrifugal dynamic filtering apparatus and cell separation system using same
KR1020147032756A KR101728939B1 (ko) 2012-12-14 2012-12-14 원심 동적 필터링 장치 및 이를 이용한 세포 분리 시스템
JP2015516411A JP5925962B2 (ja) 2012-12-14 2012-12-14 遠心動的濾過装置及びそれを用いた細胞分離システム
PCT/CN2012/086694 WO2014089838A1 (zh) 2012-12-14 2012-12-14 离心动态过滤装置及利用其的细胞分离系统
PT128900479T PT2933326T (pt) 2012-12-14 2012-12-14 Aparelho de filtração dinâmica centrífuga e sistema de separação de células utilizando o mesmo
BR112014029935A BR112014029935B1 (pt) 2012-12-14 2012-12-14 dispositivo de filtragem centrífuga e sistema de separação de células com o mesmo
US14/547,159 US20150068959A1 (en) 2012-12-14 2014-11-19 Centrifugal filtration device and cell separation system with the same
US15/230,516 US10258927B2 (en) 2012-12-14 2016-08-08 Centrifugal dynamic filtering apparatus and cell separation system using same

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JP2015519067A (ja) 2015-07-09
CN104363972A (zh) 2015-02-18
EP2933326A4 (en) 2016-09-14
RU2014144222A (ru) 2016-05-27
AU2012396668B2 (en) 2016-07-28
SG11201408169YA (en) 2015-01-29
CN104363972B (zh) 2016-08-24
BR112014029935B1 (pt) 2019-11-26
CA2873077A1 (en) 2014-06-19
CA2873077C (en) 2018-03-06
KR20150013576A (ko) 2015-02-05
BR112014029935A2 (pt) 2017-06-27
US20150068959A1 (en) 2015-03-12
KR101728939B1 (ko) 2017-04-20
JP5925962B2 (ja) 2016-05-25
PT2933326T (pt) 2021-06-17
AU2012396668A1 (en) 2014-12-11

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