JP2010220524A - Cell-treating method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-treating method, by which cell masses can more surely be disintegrated on the washing of the cells to efficiently wash the cells, thereby improving the purity of the recovered cells to maintain the cells in a healthy state. <P>SOLUTION: There is provided the cell-treating method comprising receiving a mixture liquid of a cell suspension A with a solid material B having specific gravity approximately equivalent to that of the cells to be recovered, contained in the cell suspension A, in a centrifugal container 1; and then rotating the centrifugal container 1 so that the bottom portion 1a of the container 1 is directed outward in the radial direction. Thus, when the cells are washed, the cell masses can more surely be disintegrated to effectively wash the cells, and the purity of the recovered cells can be improved to maintain the cells in a healthy state. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell treatment method.

  Conventionally, in order to collect tissue-derived cells obtained by digesting biological tissue with digestive enzymes, it is necessary to wash the digestive enzymes, and the cell suspension containing the digestive enzymes obtained in the digestion process is centrifuged. Things have been done. In this case, the supernatant obtained as a result of the centrifugation treatment is discarded, and a new washing solution is supplied, whereby the digestive enzyme concentration in the cell suspension can be reduced (see Patent Document 1). ).

International Publication No. 2007/012480 Pamphlet

  However, when cells with a large specific gravity are aggregated by applying centrifugal force, the cells adhere to each other to form a pellet-like lump, and when the washing solution is supplied next, the cell lump cannot be resuspended and cannot be resuspended. Sometimes. If resuspension is not possible, digestive enzymes and dust remain sandwiched between cells that make up the cell mass, and the purity of the recovered cells may be reduced, or the cells may be damaged. There is an inconvenience.

  The present invention has been made in view of the above-described circumstances, and at the time of cell washing, the cell mass can be released more reliably, the cells can be washed effectively, and the purity of the collected cells can be improved. It aims at providing the cell processing method which can improve and maintain a cell in a healthy state.

In order to achieve the above object, the present invention provides the following means.
The present invention contains a mixture of a cell suspension and a solid substance having a specific gravity substantially equal to that of cells to be collected contained in the cell suspension in a centrifuge container. Provided is a cell treatment method in which the bottom part is rotated so as to be directed radially outward.

  According to the present invention, when the cell suspension is accommodated in the centrifuge container and centrifuged, a solid having a specific gravity substantially equal to that of the cells to be collected is mixed in the cell suspension. The solids move radially outward with the cells to be collected. And when separating into a cell group and a supernatant, it isolate | separates in the state in which the solid substance was taken in in the cell group. Therefore, even if the cells hardened into a pellet form due to the centrifugal force during the centrifugation, the solid matter helped the disintegration of the cell mass by supplying the washing solution, and the cell mass was released and sandwiched between the cells Dust and digestive enzymes can be released. Therefore, the purity of the collected cells can be improved and healthy cells can be collected.

In the said invention, it is preferable that the said solid substance consists of a material which can be phase-transitioned between a solid phase and a liquid phase.
By doing in this way, since the solid substance which assisted the destruction of the cell mass in the washing | cleaning process becomes unnecessary in the case of collection | recovery, a cell group can be easily collect | recovered by carrying out phase transition to a liquid phase.

Moreover, in the said invention, the said solid substance may consist of a temperature sensitive material which carries out a phase transition with temperature.
In this way, after washing the cell group, by changing the temperature of the mixed solution of the washing liquid and the cell group, the solid can be phase-shifted into the liquid phase, and the cell group can be easily recovered. .

Moreover, in the said invention, the said solid substance may consist of poly-N-isopropylacrylamide.
By doing so, it becomes a solid phase at about 37 ° C., assists the disintegration of the cell mass at the time of washing, and becomes a liquid phase by cooling to 15 ° C. or less after washing, so that the cell group can be easily recovered. can do.

Moreover, in the said invention, the said solid substance may consist of the material which carries out a phase transition with a chelating agent.
In this way, after washing the cell group, by changing the pH of the mixed solution of the washing liquid and the cell group, the solid can be phase-transitioned into the liquid phase, and the cell group can be easily recovered. .

Moreover, in the said invention, the said solid substance may consist of alginate beads.
By doing this, by adding an acidic substance such as citric acid and lowering the pH, the alginate beads can be phase-transferred to the liquid phase and the collection of the cell group can be facilitated.

  In the above invention, the step of separating the mixture into a cell group and a supernatant by rotating the centrifuge container, the step of removing the layer-separated supernatant, and the residue in the centrifuge container A step of supplying a washing solution to the cell group and a step of causing the solid matter to transition to a liquid phase may be included.

  According to the present invention, during cell washing, the cell group can be released more reliably, the cells can be washed effectively, the purity of the collected cells is improved, and the cells are maintained in a healthy state. There is an effect that can be done.

It is a flowchart explaining the cell processing method which concerns on one Embodiment of this invention. In the cell processing method of FIG. 1, it is a longitudinal cross-sectional view which shows the state which accommodated the cell suspension in the centrifuge container. It is a longitudinal cross-sectional view which shows the state which performed the centrifugation step in the cell processing method of FIG. It is a longitudinal cross-sectional view which shows the supernatant removal step in the cell processing method of FIG. It is a longitudinal cross-sectional view which shows the washing | cleaning step in the cell processing method of FIG. It is a longitudinal cross-sectional view which shows the phase transition step in the cell processing method of FIG.

A cell treatment method according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the cell treatment method according to the present embodiment contains a cell suspension A containing cells to be collected and a plurality of beads (solid matter) B in a centrifuge container 1. (FIG. 2), a rotating centrifugation step S1 that rotates, and a supernatant removal step S2 that sucks and removes the supernatant D separated (FIG. 3) from the cell group C by the centrifugation step S1 (FIG. 4). ), A washing step S3 (FIG. 5) for supplying the washing liquid E after the supernatant D is removed, a centrifugation step S4 for centrifuging the cell suspension A resuspended with the washing liquid E, and the centrifugation Supernatant removal step S5 that sucks and removes the supernatant D separated from the cell group C by the separation step S4, step S6 that repeats step S3 to step S5 if necessary, and phase transition of the beads B to the liquid phase Step S7 (FIG. 6) and And a step S8 of recovering cell group C.

In the present embodiment, the beads B are made of a material having a specific gravity substantially equal to that of the cells to be collected, for example, a temperature sensitive material such as poly-N-isopropylacrylamide. Poly-N-isopropylacrylamide is phased so as to be a fluid aqueous solution (sol state) at a low temperature (0 ° C. to 15 ° C.) and a non-flowable hydrogel (gel state) at room temperature (25 ° C.) or higher. It is a material that transitions.
The concentration of beads B is 1.2% or more.

  The centrifugation steps S1 and S4 are accommodated inside as shown in FIG. 3 by rotating the centrifuge container 1 having the closed bottom portion 1a so that the bottom portion 1a is directed radially outward. The substance having a large specific gravity in the cell suspension A is moved to the bottom 1a side, and the substance having a smaller specific gravity is separated into layers.

  In the supernatant removal steps S2 and S5, the cell suspension A is separated into the cell group C at the bottom 1a and the supernatant D above it by the centrifugation steps S1 and S4, and the bottom 1a is vertically downward. Arrange to face. Then, as shown in FIG. 4, the supernatant is obtained by suctioning with the suction tube 2 in which the suction port 2 a is disposed slightly above the boundary surface F between the cell group C and the supernatant D in the centrifuge container 1. D is discharged out of the centrifuge container 1.

  In the washing step S3, as shown in FIG. 5, the washing liquid E, for example, physiological saline is supplied into the centrifuge container 1 after the supernatant D is removed in the supernatant removing steps S2 and S5. Supply from the supply port 3a. The supply port 3 a is arranged in the vicinity of the bottom 1 a of the centrifuge container 1. Thereby, the washing | cleaning liquid E can be sprayed to the cell group C deposited on the bottom part 1a of the centrifuge container 1, and the cell group C can be made easy to unravel by the momentum.

  In step S7 for causing the phase transition, for example, as shown in FIG. As a result, the cell group C in the form of pellets is resuspended by the Lactated Ringer's solution G, and the beads B that have been taken into the cell group C are phase-shifted to the liquid phase.

  According to the cell processing method according to the present embodiment, in the centrifugation steps S1 and S4, the beads B having a specific gravity substantially equal to the cells to be collected in the cell suspension A are mixed and the centrifugal force is applied. The cells move radially outward together with the beads B and are deposited and separated from the supernatant D. At this time, even if the cell group C is pressed and compressed by the centrifugal force, the contact between the cells is reduced by the beads B, so that the adhesion between the cells is reduced and the force of hardening into a pellet is weakened.

  Therefore, in the washing step S3 performed after the centrifugation steps S1 and S4, when the washing liquid E is supplied, the cell group C is easily collapsed and resuspended by the momentum of the flow of the washing liquid E.

  As a result, the cell group C is unraveled during the cleaning with the cleaning liquid E, so that dust and digestive enzymes sandwiched between the cells are released into the cleaning liquid C. Then, in the subsequent centrifugation steps S1 and S4, these dusts, digestive enzymes, etc. are separated from the cell group C and retained in the supernatant D, so that they are discharged together with the supernatant D in the supernatant removal steps S2 and S5. Is done. That is, the purity of the cells finally recovered can be improved by removing the dust, and the damage to the cells can be reduced by removing the digestive enzyme.

  Furthermore, in the cell processing method according to the present embodiment, the beads B can be phase-shifted to the liquid phase by performing step S7 of phase transition. As a result, in the subsequent recovery step S8, since the solids such as the beads B are not included in the recovered material, it can be easily aspirated and recovered by a syringe or the like.

In the present embodiment, in the phase transition step S7, the Lactated Ringer's solution G of 15 ° C. or lower is supplied, but instead, the entire container may be cooled to 15 ° C. or lower.
Further, as a material for the beads B, a temperature sensitive material such as poly-N-isopropylacrylamide which is a solid phase at room temperature and a liquid phase at low temperature is adopted. Instead, a liquid phase at high temperature such as gelatin is used. A temperature sensitive material may be used. Further, instead of the temperature sensitive material, a material in which a liquid phase and a solid phase are changed by a chelating agent, for example, alginic acid may be adopted.

  When adopting beads B made of alginic acid, the concentration is 1.2% or more, preferably 2.5% or more. Further, in step S7 for phase transition in this case, a solubilized solution composed of, for example, 55 mmol / L sodium citrate buffer (pH 6.8) is placed in the centrifuge container 1 after the supernatant D is removed. The beads B can be dissolved by supplying about 5 times the amount of beads B made of alginic acid and stirring at room temperature for 15 to 30 minutes.

1 Centrifuge container 1a Bottom A Cell suspension B Beads (solid matter)
C cell group D supernatant E washing solution

Claims (7)

  In a centrifuge container, a mixed solution of a cell suspension and a solid substance having a specific gravity substantially equal to that of the cells to be collected contained in the cell suspension is accommodated, and the bottom of the centrifuge container has a radius. A cell processing method that rotates in a direction outward.   The cell treatment method according to claim 1, wherein the solid material is made of a material capable of phase transition between a solid phase and a liquid phase.   The cell treatment method according to claim 2, wherein the solid material is made of a temperature-sensitive material that undergoes a phase transition according to temperature.   The cell treatment method according to claim 3, wherein the solid is made of poly-N-isopropylacrylamide.   The cell treatment method according to claim 2, wherein the solid material is made of a material that undergoes phase transition by a chelating agent.   The cell treatment method according to claim 5, wherein the solid material comprises alginate beads. Rotating the centrifuge container to stratify the mixture into a cell population and a supernatant; and
Removing the layered supernatant;
Supplying a washing solution to the cells remaining in the centrifuge container;
The cell treatment method according to any one of claims 2 to 6, comprising a step of causing the solid matter to undergo a phase transition to a liquid phase.

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