JP2010063441A - Methods for removing fat and treating living tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide methods for removing fat and treating a living tissue wherein the fat is efficiently removed from living tissue-derived cells that are collected from a cell suspension obtained by decomposing the living tissue. <P>SOLUTION: There is provided a method for removing fat comprising: a vessel-treating step S21 in which a surfactant solution 8 containing a surfactant 7 is brought into contact with an inner wall 6a of a condensation vessel 6 storing a cell suspension 3 obtained by decomposing the living tissue 1; emulsion-forming step S22 in which a cell suspension 3 is stirred in the condensation vessel 6; and an emulsion-removing step S23 in which an emulsion formed in the emulsion-forming step S22 is removed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a fat removal method and a biological tissue processing method.

  Conventionally, in order to obtain cells derived from living tissue, the living tissue is decomposed with a digestive enzyme solution to obtain a cell suspension containing cells separated from the living tissue, and the cell suspension is washed and concentrated to obtain desired cells. Cell concentrate (see, for example, Patent Document 1). When cells separated from living tissue are used for regenerative medicine, etc., if fat derived from tissue remains in the cell concentrate that is the final product, it may affect the therapeutic effect, so high purity cells that do not contain fat A concentrate is required.

  On the other hand, it has been reported that when pluronic F68, a surfactant, is added when stirring insect cells in a bioreactor, insect cells can grow normally without being damaged due to the protective effect of pluronic F68. (See Non-Patent Document 1).

WO05 / 012480 pamphlet David W. Murhammer & Charles F. Goochee, “Scaleup of Insect Cell Cultures: Protective Effects of Pluronic F-68”, Bio / Technology, 6, 1998, p.1411-p.1418

However, the fat contained in the cell suspension tends to be adsorbed on the inner wall of the container, and when the cell concentrate is collected after centrifugation, the fat adsorbed on the inner wall is also collected at the same time. It is difficult to obtain a liquid.
In order to prevent fat from adsorbing to the inner wall of the container, a surface treatment in which a fine uneven structure is applied to the surface of the inner wall or the surface is coated with Teflon, etc., can be considered, but there are problems that the cost is high and sterilization is weak Not practical. At the present stage, there is no ingenuity other than removing fat floating in the supernatant in order to efficiently remove fat, and there is a disadvantage that it takes time and labor to remove fat.

  The present invention has been made in view of the circumstances described above, and a fat removal method and biological tissue for efficiently removing fat from biological tissue-derived cells collected from a cell suspension obtained by decomposing biological tissue. It aims to provide a processing method.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a container processing step in which a surfactant solution containing a surfactant is brought into contact with an inner wall of a concentration container that contains a cell suspension obtained by decomposing a biological tissue, and the cell suspension in the concentration container There is provided a fat removing method comprising an emulsion forming step of stirring a liquid and an emulsion removing step of removing the emulsion formed in the emulsion forming step.

  According to the present invention, before storing the cell suspension in the concentration container, in the container processing step, the inner surface of the concentration container is contacted with the surfactant solution containing the surfactant, and the inner wall is wetted with the surfactant solution. . The inner surface of the hydrophobic concentration container is hydrophilic because the surfactant is covered with a surfactant layer arranged with the hydrophobic side facing the inner wall of the concentration container and the hydrophilic side facing the inner side in the radial direction of the concentration container. . Moreover, the state which the water droplet of surface active solution adhered to the inner wall is made. After this, the cell suspension is accommodated in a concentration container, and when the cell suspension is stirred by the emulsion formation step, the fat contained in the cell suspension forms micelles by the surfactant contained in the water droplets. These micelles are dispersed in the cell suspension to form an emulsion. Further, since the fat contained in the cell suspension has hydrophobicity, it is not adsorbed on the inner wall of the hydrophilic concentration container.

  Therefore, when the emulsion-formed solution is removed from the cell suspension in the emulsion removal step, the micellar fat is also removed at the same time. In this case, since the emulsion-formed solution is difficult to be adsorbed on the inner wall of the concentration container, the fat can be efficiently removed from the concentration container without adsorbing and remaining on the inner wall of the concentration container.

In the above invention, the surfactant solution may be a cleaning solution.
In this way, when the inside of the concentration container is rinsed with a cleaning solution containing a surfactant, the inner wall of the concentration container is covered with a layer of surfactant, and a wet state can be created. It can be performed.

In the above invention, the surfactant is preferably 2-methacryloyloxyphosphorylcholine (MPC, manufactured by NOF Corporation), Pluronic F68 (produced by ADEKA Corporation), or a perfluorocarbon emulsion.
Since MPC or Pluronic F68 or perfluorocarbon emulsion has been confirmed to be biosafety, it does not matter if it remains in the final product. In addition, it has been found that Pluronic F68 has an effect of reducing damage to cells caused by stirring.

Moreover, in the said invention, it is desirable that the said emulsion formation step stirs the said cell suspension at 5-400 rpm.
By stirring the cell suspension at 5 to 400 rpm, it is possible to efficiently form an emulsion while suppressing the generation of bubbles, and to reduce cell damage.

  Further, the present invention is obtained by a tissue degradation step of degrading a living tissue with a digestive enzyme solution to obtain a cell suspension, a fat removal step of removing fat from the cell suspension, and the fat removal step A cell washing step for washing and concentrating cells derived from biological tissue, wherein the fat removal step comprises contacting a surfactant solution containing a surfactant with an inner wall of a concentration vessel containing the cell suspension; There is provided a biological tissue treatment method comprising an emulsion forming step of stirring the cell suspension in the concentration container, and an emulsion removing step of removing the emulsion formed in the emulsion forming step.

  According to the present invention, when a biological tissue is agitated in a digestive enzyme solution containing a digestive enzyme in the tissue decomposition step to decompose the biological tissue, the biological tissue-derived cells are separated from the biological tissue to obtain a cell suspension. Since the fat content derived from living tissue is contained in the cell suspension, the fat content is removed from the cell suspension in the fat removal step to obtain a cell concentrate of living tissue-derived cells containing almost no fat content. In the cell washing step, the cell concentrate obtained in the fat removal step is stirred in the washing solution, and then separated into a supernatant mixed with precipitates and impurities of biological tissue-derived cells by centrifugation, and the supernatant is removed. Thus, a cell concentrate of washed biological tissue-derived cells, which is the final product, can be obtained.

  In this case, in the fat removal step, before the cell suspension obtained in the tissue decomposition step is accommodated in the concentration container, the container treatment step is performed in advance so that the inner wall of the concentration container is brought into contact with the surfactant-containing surfactant. Keep it. Thus, when the cell suspension is agitated in the emulsion formation step, the fat content forms micelles, and these micelles are dispersed in the cell suspension to form an emulsion. Minutes can be removed. Further, at this time, the fat is not adsorbed and remains on the inner wall of the concentration container, so that the fat can be efficiently removed.

  Also in the subsequent cell washing step, the fat remaining in the cell concentrate becomes micelle by the remaining surfactant and is dispersed in the washing liquid, so that the fat remaining simultaneously with the washing liquid can also be removed. Further, since the inner wall of the concentration container covered with the surfactant layer in the container processing step is kept hydrophilic, it is possible to prevent fat from being adsorbed on the inner wall when removing the cleaning liquid. As a result, there is no inconvenience of collecting fat at the same time when the washing liquid is discharged and the cell concentrate of the living tissue-derived cells remaining in the concentration container is recovered, and high-quality living tissue-derived cells are used as the final product. Obtainable.

In the above invention, after the container processing step, a tissue decomposition step may be performed in a concentration container in which the surface active solution is brought into contact with the inner wall in the container processing step.
By doing in this way, when the inner wall surface is covered with the surfactant layer and becomes hydrophilic, and the tissue decomposition step is performed in the concentration container with the water droplets of the surface active solution attached, the biological tissue is decomposed, The fat separated from the living tissue can be micellar and dispersed in the digestive enzyme solution to form an emulsion.

  That is, since the tissue decomposition step and the emulsion formation step can be performed at the same time, the work efficiency increases. Moreover, since the agitation performed in each of the tissue degradation step and the emulsion formation step can be reduced to one time, damage to cells due to agitation can be reduced.

In the above invention, the surfactant solution may be a cleaning solution.
Moreover, in the said invention, it is preferable that the said surfactant is MPC, pluronic F68, or a perfluorocarbon emulsion.
Moreover, in the said invention, it is preferable that the said emulsion formation step stirs a cell suspension at 5-400 rpm.

  According to the present invention, there is an effect that fat can be efficiently removed from living tissue-derived cells collected from a cell suspension obtained by decomposing living tissue.

A fat removal method and a biological tissue processing method according to an embodiment of the present invention will be described below with reference to FIG.
As shown in FIG. 1, the biological tissue processing method according to this embodiment includes a tissue decomposition step S1 in which a biological tissue 1 is decomposed with a digestive enzyme solution 2 to obtain a cell suspension 3, and a tissue decomposition step S1. A fat removing step S2 for removing the fat 4 derived from the living tissue 1 from the obtained cell suspension 3 and a cell washing step S3 for washing and concentrating the living tissue-derived cells 5 from which the fat 4 has been removed are provided. .
The fat removal method according to the present embodiment is the fat removal step S2.

  The lipolysis step S1 is performed by stirring the living tissue 1 in the digestive enzyme solution 2. When the biological tissue 1 is decomposed by the digestive enzyme, the biological tissue-derived cells 5 are separated from the biological tissue 1, and a cell suspension 3 in which the biological tissue-derived cells 5 float in the digestive enzyme solution 2 is obtained.

  The fat removal step S2 includes a container processing step S21 in which the inner wall 6a of the concentration container 6 is brought into contact with the surfactant solution 8 containing the surfactant 7, and the cell suspension 3 in the concentration container 6 in contact with the surfactant solution 8. Are formed, and an emulsion formation step S22 for forming an emulsion and an emulsion removal step S23 for removing the emulsion formed in the emulsion formation step S22 are provided.

  The container processing step S21 is, for example, a 1% pluronic F68 / LR solution (surfactant solution 8) that is a 1% pluronic F68 / LR solution obtained by adding 1% surfactant pluronic F68 to a lactic acid Ringer solution (hereinafter referred to as LR solution) that is a cleaning solution 9. 6 by rinsing the interior. As a result, the inner wall 6a of the hydrophobic concentration container 6 has the pluronic F68 lined up with the pluronic F68 aligned with the hydrophobic side toward the inner wall 6a side of the concentration container 6 and the hydrophilic side toward the radially inner side of the concentration container 6. It is covered with a layer and becomes hydrophilic.

  Further, the inner wall 6a is in a state where water droplets of 1% pluronic F68 / LR liquid are attached. The emulsion formation step S22 is performed by storing the cell suspension 3 obtained in the tissue decomposition step S1 in such a concentration container 6 and stirring at 5 to 400 rpm. The fat 4 derived from the living tissue 1 contained in the cell suspension 3 becomes micelles by the Pluronic F68 attached to the inner wall 6a of the concentration container 6, and is dispersed into the cell suspension 3 to form an emulsion.

  Then, the concentration container 6 is centrifuged by emulsion removal step S23, and it isolate | separates into the sediment containing the biological tissue origin cell 5 and the supernatant which formed the emulsion. When the supernatant is removed, the fat 4 dispersed in a micelle form in the supernatant is also removed, and a cell concentrate of the biological tissue-derived cells 5 containing almost no precipitated fat 4 remains in the concentration container 6.

  In the cell washing step S3, the washing solution 9, for example, LR solution, is injected into the concentration vessel 6 in which the cell concentrate obtained in the emulsion removal step S23 remains, and the mixture is stirred and centrifuged. It is carried out by separating into a supernatant of the liquid and removing the supernatant. Fat 4 remaining in the cell concentrate forms micelles by pluronic F68 remaining in the cell concentrate, is dispersed in the LR solution, and is discharged together with the LR solution.

  The remaining impurities other than fat 4 are also dispersed in the LR liquid by stirring and discharged together with the LR liquid. By repeating this cell washing step S3 one or more times, impurities remaining in the cell concentrate are removed, and a cell concentrate of the biological tissue-derived cells 5 as the final product is obtained.

  In this case, since the inner wall 6a of the concentration container 6 covered with the layer of Pluronic F68 in the container processing step S21 is kept hydrophilic, the fat 4 remaining in the cell concentrate is discharged from the inner wall when the LR solution is discharged. 6a is not adsorbed. As a result, when the cell concentrate of the biological tissue-derived cells 5 that is the final product washed with the LR solution is recovered from the concentration container 6, the fat 4 adsorbed on the inner wall 6a of the concentration container 6 is simultaneously recovered. There is an advantage that a cell concentrate of the biological tissue-derived cell 5 having a high purity and free from fat 4 can be obtained.

  As described above, according to the fat removal method and the biological tissue processing method according to the present embodiment, the cell suspension 3 accommodated in the concentration container 6 simply by washing the inside of the concentration container 6 with the pluronic F68 / LR solution. The fat 4 derived from the living tissue 1 can be dispersed therein to form an emulsion, and the fat 4 can be prevented from adsorbing to the inner wall 6a of the concentration container 6. As a result, the fat content 4 can be efficiently removed from the living tissue-derived cells 5, and the time and labor required for the processing of the living tissue 1 can be reduced.

  In the present embodiment, after the container processing step S21, the tissue decomposition step S1 is performed in the concentration container 6 in which the surface active solution 8 is brought into contact with the inner wall 6a of the concentration container 6 in the container processing step S21. Also good.

  In the container processing step S21, the inner wall 6a of the concentration container 6 is covered with the Pluronic F68 layer and becomes hydrophilic, and the living tissue 1 and the digestive enzyme solution 2 are formed in the concentration container 6 in which water droplets of the Pluronic F68 / LR liquid are attached. And the biological tissue 1 is decomposed by stirring. By doing in this way, the process of decomposing | disassembling the biological tissue 1 and the process which the fat part 4 contained in the biological tissue 1 becomes a micelle form, and it disperse | distributes in the digestive enzyme solution 2, and can form an emulsion can be performed simultaneously. , Can increase the efficiency of work. Moreover, since the stirring process can be reduced, damage to the living tissue-derived cells 5 can be reduced.

In the present embodiment, Pluronic F68 is used as the surfactant 7, but MPC or perfluorocarbon emulsion may be used instead.
Pluronic F68 or MPC or perfluorocarbon emulsion has been confirmed to be biosafety, and even if it remains in the final product, there is no problem with safety to the living body.

  In the present embodiment, the cell suspension 3 is stirred at 5 to 400 rpm in the emulsion forming step S22, but it is desirable to stir at 30 to 100 rpm. By doing in this way, the damage to the biological tissue origin cell 5 in the cell suspension 3 can be reduced, forming an emulsion efficiently.

  Moreover, in this embodiment, in the emulsion removal step S23, the concentration container 6 is centrifuged to separate the biological tissue-derived cell 5 from the precipitate and the emulsion supernatant, and the supernatant is removed. The emulsion may be removed from the concentration container 6 by a filter.

  In the present embodiment, the 1% pluronic F68 / LR solution is injected in the container processing step S21. Instead of this, the pluronic F68 and the LR solution are independently injected, and 1 in the concentration container 6 is injected. % Pluronic F68 / LR solution may be prepared

Moreover, in this embodiment, if the fat 4 derived from the biological tissue 1 becomes a micelle shape and forms an emulsion by Pluronic F68, the emulsion does not need to be separated.
Emulsions can be used as nutrients in the arteries.

It is a conceptual diagram which shows the procedure of the fat removal method and biological tissue processing method which concern on one Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Living tissue 2 Digestive enzyme solution 3 Cell suspension 4 Fat content 5 Living tissue origin cell 6 Concentration container 6a Inner wall 7 Surfactant 8 Surfactant 9 Washing solution S1 Tissue decomposition step S2 Fat removal step S21 Container processing step S22 Emulsion formation Step S23 Emulsion removal step S3 Cell washing step

Claims (9)

A container treatment step in which a surfactant solution containing a surfactant is brought into contact with an inner wall of a concentration container containing a cell suspension obtained by decomposing a biological tissue;
An emulsion forming step of stirring the cell suspension in the concentration vessel;
A fat removal method comprising: an emulsion removal step of removing the emulsion formed in the emulsion formation step.   The fat removal method according to claim 1, wherein the surfactant solution is a cleaning solution.   The fat removal method according to claim 1 or 2, wherein the surfactant is MPC, Pluronic F68, or perfluorocarbon emulsion.   The fat removal method according to any one of claims 1 to 3, wherein the emulsion forming step stirs the cell suspension at 5 to 400 rpm. A tissue degradation step of degrading a biological tissue with a digestive enzyme solution to obtain a cell suspension;
Removing fat from the cell suspension; and
A cell washing step of washing and concentrating cells derived from the biological tissue obtained in the fat removal step,
The fat removal step comprises:
A container treatment step in which a surfactant solution containing a surfactant is brought into contact with an inner wall of a concentration container containing the cell suspension; and
An emulsion forming step of stirring the cell suspension in the concentration vessel;
A biological tissue treatment method comprising: an emulsion removal step of removing the emulsion formed in the emulsion formation step.   The biological tissue processing method according to claim 5, wherein after the container processing step, the tissue decomposition step is performed in a concentration container in which a surface-active solution is brought into contact with an inner wall in the container processing step.   The biological tissue treatment method according to claim 5 or 6, wherein the surface-active solution is a cleaning liquid.   The biological tissue treatment method according to any one of claims 5 to 7, wherein the surfactant is MPC, pluronic F68, or perfluorocarbon emulsion.   The biological tissue treatment method according to any one of claims 5 to 8, wherein the emulsion forming step stirs the cell suspension at 5 to 400 rpm.

Images