JPWO2006109367A1 - Cell release method, cell release solution, cell culture method, cell culture solution, cell solution, cell solution preparation, cell fixing method and cell fixing solution - Google Patents

Abstract

An object of the present invention is to provide a method in which adherent animal cells are normally cultured and proliferated in a floating state, and the cultured and proliferated adherent animal cells are re-established in a desired place. A cell release method according to the present invention is characterized in that lactoferrin is added to a culture solution of a culture vessel in which cells are cultured. In addition, the cell culture method according to the present invention is characterized in that cells are suspended and cultured in a culture solution containing lactoferrin. The cell fixing method according to the present invention is characterized by diluting lactoferrin, transferring a culture solution containing floating cells to a laminin-coated incubator, or adding glycosaminoglycan. In these, the cells are adherent animal cells, and the culture medium contains serum. [Selection] Figure 1

Description

  The present invention relates to a cell release method, a cell release solution, a cell culture method, a cell culture solution, a cell solution, a cell solution preparation, a cell fixing method and a cell fixing solution using lactoferrin.

  Recent advances in medical technology and testing technology are remarkable, and new medical technology and new testing technology have been developed one after another. In the development of new medical techniques and new testing techniques, various experiments and research using cells have been conducted.

  In various experiments and research using cells, it is necessary to prepare the necessary number of cells and number of culture dishes according to the purpose, so it is necessary to continuously culture untreated cells (master cells) continuously. I must.

  If necessary, the master cells that have been continuously subcultured are detached from the culture dish and suspended if necessary, and part of the cells are again transferred to the master cell culture dish for subculture, and the cell suspension The rest is sowed and grown on the culture dish in the appropriate size and the necessary number while calculating the growth rate so that the number of cells required for the experiment / research will be met by the time of the experiment. .

  It is not impossible to wake up cells that have been frozen and stored in liquid nitrogen each time without culturing the master cells, but to prepare the cells for use in experiments. Since it takes too much time, the subculture method as described above is usually employed.

  Cells grown to the desired number of cells are used according to the content of the experiment and research. Here, the proliferated cells may be used while adhered on the culture dish or may be used after being detached from the culture dish.

  When detaching from the culture dish, the cells must be detached from the culture dish.However, as a method of detaching the cells from the culture dish, generally, a protease such as trypsin is added to the culture solution, A method is used in which cells are detached by dissolving the joint of the cultured cells with the culture dish. And in this method, the method of peeling a cell by spraying the liquid flow of a culture solution on the cell to peel and flushing may be taken together.

  However, if a proteolytic enzyme such as trypsin is used or flushing is performed by spraying a liquid flow of a culture solution, there is a problem that the cultured cells are damaged and it takes time to recover their functions.

  Moreover, there are cases where the cultured cells have to be transported to another place due to various circumstances. Since cultured cells die when they are detached from the inner surface of the incubator, the culture solution in the flask in which the cells are cultured is shaken during transportation so that the cultured cells are not detached and the flask is further filled with the culture solution. Is trying not to shake.

However, even if such measures are taken, transportation of cultured cells is very troublesome because they must be transported promptly and carefully without applying shocks such as vibration.
JP 2004-75547 A JP-A-8-66184

  The problem to be solved is that a method for culturing and growing adherent animal cells normally in a floating state and re-establishing the cultured and grown adherent animal cells at a desired location has not been established.

  The cell release method according to the present invention is performed by culturing adherent animal cells in a cell culture solution while adhering to the surface of the carrier, and then mixing the cell release solution containing lactoferrin with the cell culture solution. Cell release method for releasing the adherent animal cells from the serum, wherein the cell culture solution contains serum in a concentration range of 5 to 20%, and the cell release solution has a concentration of lactoferrin when mixed with the cell culture solution. Is characterized by containing lactoferrin at a concentration of 10 to 500 μM.

  Here, the reason that the serum concentration is 5 to 20% is that when the serum concentration is less than 5% or exceeds 20%, there is a disadvantage that the cell growth rate decreases. The reason why the concentration of lactoferrin is 10 to 500 μM is that when the concentration of lactoferrin is less than 10 μM, the cell release effect is small, and when the concentration exceeds 500 μM, the cell release effect is saturated.

  Moreover, it is preferable that the component except the lactoferrin of the said cell free solution is substantially the same as the component of the said cell culture solution at the point that the released cell can be cultured as it is.

  Moreover, the cell free solution according to the present invention is characterized by comprising a cell culture solution containing at least 1 to 10 mM of lactoferrin. Here, the reason why the concentration of lactoferrin in the culture solution is 1 to 10 mM is that if the concentration of lactoferrin is within this range, it is easy to use.

  The cell culture method according to the present invention is characterized by culturing adherent animal cells in a suspended state in a cell culture solution containing 5 to 20% serum and 10 to 500 μM lactoferrin. Here, the concentration of lactoferrin in the culture solution is preferably 10 to 500 μM. The reason why the concentration of lactoferrin is set to 10 to 500 μM is that if the concentration of lactoferrin is less than 10 μM, the effect of suspension culture of the cells is reduced and the effect is fixed, and if the concentration of lactoferrin exceeds 500 μM, the effect of suspension culture of the cells is saturated. It is.

  The cell culture medium according to the present invention is characterized by containing 5 to 20% serum and 10 to 500 μM lactoferrin. Here, the reason that the serum concentration is 5 to 20% is that when the serum concentration is less than 5% or exceeds 20%, there is a disadvantage that the cell growth rate decreases. In addition, the concentration of lactoferrin in the culture solution is set to 10 to 500 μM because if the concentration of lactoferrin is less than 10 μM, the effect of suspension culture of the cells is reduced and the effect is fixed. If the concentration of lactoferrin exceeds 500 μM, the effect of suspension culture of the cells is achieved. Is saturated.

  The cell solution according to the present invention is characterized by comprising a cell culture solution containing 5 to 20% serum, 10 to 500 μM lactoferrin, and adhering animal cells in a floating state. Here, the reason that the serum concentration is 5 to 20% is that when the serum concentration is less than 5% or more than 20%, there is a disadvantage that the cell viability decreases. In addition, the concentration of lactoferrin in the culture solution is set to 10 to 500 μM because when the concentration of lactoferrin is less than 10 μM, the effect of floating the cells decreases and the effect of floating the cells is saturated, and when the concentration exceeds 500 μM, the effect of floating the cells is saturated. Because it will do.

  Moreover, the cell fluid preparation according to the present invention comprises a syringe barrel and a cell fluid filled in the syringe barrel, and the cell fluid contains 5 to 20% serum and 10 to 500 μM lactoferrin, and further adheres. It is characterized by containing sex animal cells in a suspended state. Here, the reason that the serum concentration is 5 to 20% is that when the serum concentration is less than 5% or more than 20%, there is a disadvantage that the cell viability decreases. In addition, the concentration of lactoferrin in the culture solution is set to 10 to 500 μM because when the concentration of lactoferrin is less than 10 μM, the effect of floating the cells decreases and the effect of floating the cells is saturated, and when the concentration exceeds 500 μM, the effect of floating the cells is saturated. Because it will do.

  In the cell fixing method according to the present invention, a cell solution containing adhering animal cells in a floating state and lactoferrin is placed in a fixed part, and then a culture solution not containing lactoferrin is added to the fixed part. Cell fixing method in which the concentration of lactoferrin is reduced and the adherent animal cells are fixed in the part to be fixed, wherein the cell fluid contains 5 to 20% serum and 10 to 500 μM lactoferrin. It is.

  Here, the concentration of lactoferrin in the cell solution reduced in concentration by a culture solution not containing lactoferrin is preferably less than 10 μM. This is because colonization of floating cells is remarkable at less than 10 μM.

  In another cell fixing method according to the present invention, a cell solution containing floating adherent animal cells is placed in a planned fixing portion, and then the floating effect of the adherent animal cells is abolished in the cell fluid of the planned fixing portion. A cell fixing method in which the adherent animal cells are fixed to the planned fixing portion by adding a revoking substance to be fixed, wherein the cell fluid contains 5 to 20% serum and 10 to 500 μM lactoferrin It is.

  Here, as the cell fixing solution, a solution containing glycosaminoglycan in the culture solution can be used. Further, the concentration of glycosaminoglycan in the cell fluid when the adhering animal cells in a floating state are fixed is preferably 10 to 500 μg / ml. This is because if the concentration of glycosaminoglycan is less than 10 μg / ml, the effect of fixing floating cells is insufficient, and if it exceeds 500 μg / ml, the effect of fixing floating cells is saturated.

  The cell fixing solution according to the present invention is characterized in that the culture solution contains glycosaminoglycan.

  Further, according to another cell fixing method of the present invention, a fixing substance for fixing an adherent animal cell is coated on the planned fixing portion, and then a cell solution containing the floating adherent animal cell is put in the fixed fixing portion. A cell fixing method in which the adherent animal cells are fixed on the part to be fixed, wherein the fixing substance is an extracellular matrix protein, and the cell fluid contains at least 5 to 20% serum and 10 to 500 μM lactoferrin. It is characterized by this.

  Here, an extracellular matrix protein can be used as the terminator. As the extracellular matrix protein, for example, laminin can be used when floating cells are nerve cells, and fibronectin or collagen can be used when floating cells are other cells such as epithelial cells and fibroblasts. it can.

  In each of the above inventions, the culture vessel refers to all vessels that can hold and culture cells, such as culture dishes and culture flasks. In each of the above inventions, the adherent animal cells refer to, for example, nerve cells, glial cells, epithelial cells, various undifferentiated cells (mesenchymal cells, stem cells, etc.), cancer cells and the like. In each of the above inventions, the culture solution includes not only those containing nutrients for growing cells but also those that simply hold cells such as physiological saline.

  According to the present invention, there is an effect that cultured animal animal cells can be detached from a carrier without damage. Moreover, since it is not necessary to restore the function of the cultured cells that have been released over time, there is an effect that it is possible to quickly obtain cultured cells that are not damaged.

  In addition, according to the present invention, since the cell proliferation ability is higher than that of the cell to which the floating cell is adhered, and the cell can be normally cultured in the floating state, There is an effect that it can be obtained quickly and in large quantities.

  Further, according to the present invention, since the cells can be cultured in a floating state, there is an effect that the cultured cells can be easily transported and transported.

  In addition, according to the present invention, a simple method of diluting lactoferrin, transferring a culture solution to a laminin-coated incubator, or adding glycosaminoglycan can be performed from a floating cell to a fixed cell. Can be easily obtained.

  The purpose of culturing and growing adherent animal cells in a floating state and re-establishing them in a desired place was realized by using lactoferrin and glycosaminoglycan.

  Here, lactoferrin is a protein with a molecular weight of about 80 kD found in milk, has various sugar chain structures and affinity, has the property of binding to cell surface proteins and glycosaminoglycans, It has attracted attention for its various effects such as suppression of microbial infections such as viruses and protozoa, and anti-inflammatory action.

  The present inventor added bovine lactoferrin (bLf) to a medium of rat adrenal medullary pheochromocytoma-derived PC12 cells, which is widely used in the study of nerve cells, for the purpose of examining the effects of lactoferrin on neurons. Investigated about.

Prepare plurality of culture dishes in culture medium, plated PC12 cells culture dish dish per 7 × 10 ^4, the PC12 cells, 5% CO ₂ atmosphere, for 72 hours at 37 ° C.. Here, DMEM (Dulbecco's Modified Eagle Medium) medium {5% (v / v) unheated horse serum, 5% (v / v) unheated fetal bovine serum, 1 × 10 ⁵ units / l penicillin G Sodium 1 × 10 ⁵ mg / l streptomycin sulfate, 0.044M NaHCO ₃ } was used, and PC12 cells were used from RIKEN Cell Development Bank.

  After culturing for 72 hours, bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 0 μM to 500 μM. Here, bovine lactoferrin (bLf) added to the culture solution was diluted beforehand with the culture solution. The concentration of bovine lactoferrin (bLf) in the diluted solution is easily 1 to 10 mM. Note that lactoferrin is not diluted with a culture solution, but may be diluted with a solution containing no nutrients such as physiological saline.

  24 hours after adding bovine lactoferrin (bLf), the culture solution in the incubator was observed with a microscope, and floating PC12 cells were observed. The absorbance (OD) of this culture solution at a wavelength of 570 nm was measured by a cell activation evaluation test (MTT assay), and the number of cells released in the culture solution {the number of cells is proportional to the absorbance (OD). It was as shown in the graph of FIG.

  From the results shown in FIG. 1, it can be seen that bovine lactoferrin (bLf) has the effect of inhibiting the adhesion of PC12 cells onto the culture dish and freeing and floating the cells when the concentration exceeds 10 μM. Further, the number of cells floating in the culture medium increases as the concentration of bovine lactoferrin (bLf) increases, but it can be seen that the action is saturated when the concentration exceeds 500 μM.

  Since bovine lactoferrin (bLf) is charged with a positive charge at a molecular weight of about 80 kD, it is possible that these physical properties may have the above effects. Therefore, in order to confirm that the above action is specific to bovine lactoferrin (bLf), bovine serum albumin (67 kD) which is a protein having a large molecular weight in the same manner as bovine lactoferrin (bLf) instead of bovine lactoferrin (bLf). ) And lysozyme (14 kD), which is a positively charged protein, were also examined in the same manner as bovine lactoferrin (bLf).

  However, no action was observed on the bovine serum albumin (67 kD) and lysozyme (14 kD) to release cells from the culture dish as observed with bovine lactoferrin (bLf). Therefore, it can be seen that the action of releasing PC12 cells from the culture dish is specific to bovine lactoferrin (bLf) and is not caused by high molecular weight or charge.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture medium in the culture dish so that the concentration in the culture medium was 100 μM, and further cultured for 12 hours, with a wavelength of 570 nm. The absorbance (OD) of this culture solution was measured with the MTT assay as 0.5h, 1h, 3h, 6h, 12h over time, and the number of cells released in the culture solution {the number of cells is the absorbance ( Which is proportional to OD) was examined every hour. The result was as shown in FIG.

  From the results shown in FIG. 2, under the condition of bovine lactoferrin (bLf) concentration of 100 μM, the cells that had settled on the bottom of the culture dish were released a little from 0.5 h by the action of bovine lactoferrin (bLf). It can be seen that it is quite liberated in 3h and almost free in 6h.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours. Through this process, the cells proliferating in a state of being fixed on the inner surface of the culture dish are released from the inner surface of the culture dish and float in the culture solution.

  Next, cells floating in the culture medium are collected, and a culture dish prepared in advance, that is, a culture dish {bLf (+)} in which bovine lactoferrin (bLf) is present at a concentration of 100 μM in the culture liquid, The collected cells are transferred to a culture dish {bLf (−)} in which bovine lactoferrin (bLf) is not present in the culture medium, and further cultured for up to 144 hours. The absorbance (OD) of this culture liquid at a wavelength of 570 nm is measured with MTT. The number of cells released in the culture broth (the number of cells is proportional to the absorbance (OD)) was determined by measurement over time. The result was as shown in FIG.

  From the results shown in bLf (+) in FIG. 3, it is found that the floating cells proliferate with time in a floating state in an environment where the concentration of bovine lactoferrin (bLf) is 100 μM.

  Further, from the results shown in bLf (+) and bLf (−) in FIG. 3, the absorbance (OD) of the culture solution {bLf (+)} containing bovine lactoferrin (bLf) and the culture solution containing no bovine lactoferrin (bLf). When the absorbance (OD) of {bLf (-)} is compared, the absorbance (OD) of the culture solution {bLf (+)} containing bovine lactoferrin (bLf) is high, that is, the culture solution containing bovine lactoferrin (bLf) It can be seen that the cells in {bLf (+)} have a higher cell growth ability than the cells in the culture solution {bLf (−)} not containing bovine lactoferrin (bLf).

  Moreover, a culture solution (hereinafter referred to as “cell solution”) in which floating cells are cultured is filled into a syringe, and this is held at 37 ° C. for 12 hours. Thereafter, the absorbance (OD) of this culture solution at a wavelength of 570 nm is measured. When the number of cells released in the cell fluid (the number of cells is proportional to the absorbance (OD)) was measured by MTT assay, the cells were alive and the number was increased by proliferation. It increased 1.1 times compared with before the retention.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours.

  Next, the cells floating in the culture solution were collected, transferred to each culture dish containing culture solutions having bovine lactoferrin (bLf) concentrations of 0 μM, 100 μM, 300 μM, 500 μM, and 600 μM, and cultured for 24 hours. Then, the absorbance (OD) of the culture medium in each culture dish at a wavelength of 570 nm was measured by MTT assay, and the number of cells floating in the culture liquid of each culture dish {the number of cells was proportional to the absorbance (OD). I checked}. The result was as shown in FIG.

  From the results shown in FIG. 4, it can be seen that when the concentration of bovine lactoferrin (bLf) in the culture solution is increased, the proliferation ability of the cells is increased.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours.

  Next, the culture solution that has become suspended cells by the addition of bovine lactoferrin (bLf) is transferred to another culture dish, to which a culture solution that does not contain bovine lactoferrin (bLf) is added, and bovine lactoferrin ( The concentration of bLf) was reduced to 10 to 100 μM, and the mixture was further allowed to pass at 37 ° C. for 24 hours. Through this process, it was visually observed that cells were fixed on the bottom of the culture dish.

  Next, the culture solution in the culture dish is discarded, the inner surface of the culture dish is washed with a washing solution, and the absorbance (OD) of this washing solution at a wavelength of 570 nm is measured for each culture dish by MTT assay, and the cells released in the washing solution. The number of cells {the number of cells is proportional to the absorbance (OD)} was as shown in FIG.

  From the results shown in FIG. 5, when the concentration of bovine lactoferrin (bLf) in the culture solution is reduced by diluting with a diluent not containing bovine lactoferrin (bLf), the cells suspended in the culture solution are removed from the inner surface of the culture dish. It can be seen that it settles again.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours.

  Next, the culture solution containing suspended cells by the addition of bovine lactoferrin (bLf) is transferred to another culture dish, and the culture solution contains glycosaminoglycans such as heparin and chondroitin sulfate A and C. (Cell fixing solution) was added in the range of 1 to 500 μg / ml, and further allowed to pass for 24 hours. It was visually observed that PC12 cells were fixed on the bottom of the culture dish during this process.

  Next, the absorbance (OD) of the culture solution in this culture dish at a wavelength of 570 nm was measured for each culture dish by MTT assay, and the number of cells released in the culture solution {the number of cells is the absorbance (OD). It is as shown in FIG.

  From the results shown in FIG. 6, when a culture solution (cell fixing solution) containing glycosaminoglycan such as heparin or chondroitin sulfate is added, the number of cells suspended in the culture solution is reduced. It was found that the cells that had been suspended in the colony settled again on the inner surface of the culture dish.

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours.

  Next, the culture medium that contains floating cells by the addition of bovine lactoferrin (bLf) is prepared by using a pre-prepared laminin-coated culture dish (Lam +) and a laminin-coated culture dish. (Lam-) and further 24 hours passed.

  Next, the absorbance (OD) of the culture solution in this culture dish at a wavelength of 570 nm was measured over time by MTT assay, and the number of cells released in the culture solution {the number of cells was proportional to the absorbance (OD). I checked}. The result was as shown in FIG.

  Further, when the inner surface of the culture dish was observed, it was confirmed that PC12 cells were fixed and proliferated on the inner surface of the culture dish. From the above, it can be seen that the cells suspended in the culture solution are fixed to the bottom of the culture dish by the action of laminin in the presence of bovine lactoferrin (bLf).

  PC12 cells were cultured for 72 hours in the same manner as in Example 1, and then bovine lactoferrin (bLf) was added to the culture solution in the culture dish so that the concentration in the culture solution was 100 μM, and allowed to pass for 24 hours.

  Next, the culture medium containing suspended cells by the addition of bovine lactoferrin (bLf) was prepared by using a previously prepared laminin-coated culture dish (Lam +) or fibronectin-coated culture dish (Lam). -), Moved to a non-coat culture dish (non-coat), further cultured for 24 hours, and the absorbance (OD) of the culture medium in this culture dish at a wavelength of 570 nm was measured for each culture dish by MTT assay. Then, the number of cells released in the culture solution (the number of cells is proportional to the absorbance (OD)) was examined.

  The results are as shown in FIG. 8. The floating cells in the culture medium in the culture dish coated with laminin are greatly reduced, and the floating cells in the culture liquid not coated with laminin are reduced. I did not. Further, when the inner surface of the culture dish was observed, it was confirmed that PC12 cells were fixed and proliferated on the inner surface of the culture dish.

  Despite the presence of 100 μM bovine lactoferrin (bLf), the floating cells transferred to the laminin-coated culture dish decreased in number, and the number of cells that grew on the bottom of the culture dish increased (bLf +, Lam +). However, the floating cells transferred to the fibronectin-coated culture dish maintained the floating state and developed (bLf +, FN +). Therefore, it can be seen that the adhesion inhibitory effect of bovine lactoferrin (bLf) on neurons is ineffective for integrin-dependent cell adhesion formed between integrins expressed in neurons and laminin.

  In order to confirm that bovine lactoferrin (bLf) acts directly on PC12 cells, biotinylated bLf (b-bLf) was added to PC12 cells, and after 24 hours, all cells were collected and washed. The cells were solubilized, subjected to SDS polyacrylamide electrophoresis, and blotted onto a nitrogen cellulose membrane, and then analyzed for b-bLf binding. The electrophoretic pattern was as shown in FIG.

  In FIG. 9, (a) is treated with b-bLf alone, (b) is b-bLf + bLf 100 μM, (c) is b-bLf + bLf 500 μM, (d) is b-bLf + BSA 100 μM, (e) is b -BLf + BSA 500 μM.

  As a result of the analysis, b-bLf was detected, and it was found that b-bLf was directly acting on PC12 cells.

  By elucidating research on means for controlling the mechanism of cell floating and colonization, it may be applicable to the regeneration of living tissues of humans and animals.

It is a graph which shows the relationship between the density | concentration (micromol) of the bovine lactoferrin (bLf) in a culture solution, and the light absorbency (O.D.) of a culture solution. 2 is a graph showing the relationship between the elapsed time after adding bovine lactoferrin (bLf) to the culture solution and the absorbance (O.D.) of the culture solution. It is a graph which shows the relationship between the time after starting culture | cultivation of the collect | recovered cell in another culture dish culture solution, and light absorbency (O.D.). It is a graph which shows the relationship between the density | concentration (micromol) of the bovine lactoferrin (bLf) in a culture solution, and the light absorbency (O.D.) of a culture solution. It is a graph which shows the relationship between the density | concentration (micromol) of the bovine lactoferrin (bLf) in a culture solution, and the light absorbency (O.D.) of a culture solution. It is a graph which shows the relationship between the addition amount (microgram / ml) of glycosaminoglycan, and the light absorbency (O.D.) of a culture solution. It is a graph which shows the relationship between the presence or absence of laminin, and the light absorbency (O.D.) of a culture solution. It is a graph which shows the relationship between the presence or absence of laminin and the presence or absence of fibronectin, and the light absorbency (O.D.) of a culture solution. It is a figure which shows the electrophoretic pattern of b + bLf when PC12 cell cultured in b-bLf and bLf of each density | concentration presence or BSA is wash | cleaned and electrophoresed.

Claims (15)

  Culturing the adherent animal cells in the cell culture medium in a state of adhering to the surface of the carrier, and then mixing the cell free solution containing lactoferrin and the cell culture solution to release the adherent animal cells from the carrier. In the cell release method, the cell culture solution contains serum in a concentration range of 5 to 20%, and the cell release solution has a lactoferrin concentration at a concentration of 10 to 500 μM when mixed with the cell culture solution. A cell release method comprising:   The cell release method according to claim 1, wherein the components of the cell free solution excluding lactoferrin are substantially the same as the components of the cell culture solution.   A cell free solution comprising a cell culture solution containing 5 to 20% serum and 1 to 10 mM lactoferrin.   A cell culture method characterized by culturing adherent animal cells in a suspended state in a cell culture medium containing 5 to 20% serum and 10 to 500 µM lactoferrin.   A cell culture medium comprising 5 to 20% serum and 10 to 500 μM lactoferrin.   A cell solution comprising a cell culture solution containing 5 to 20% serum, 10 to 500 μM lactoferrin and containing adherent animal cells in a suspended state.   It is composed of a syringe barrel and a cell fluid filled in the syringe barrel, and the cell fluid contains 5 to 20% serum, 10 to 500 μM lactoferrin, and further contains adherent animal cells in a floating state. A cell fluid preparation.   A cell solution containing floating adhering animal cells and lactoferrin is placed in the planned fixing part, and then a cell culture solution not containing lactoferrin is added to the planned fixing part to reduce the concentration of lactoferrin in the cell liquid, and the fixing planned A cell fixing method for fixing the adherent animal cells to a part, wherein the cell solution contains 5 to 20% of serum and 10 to 500 μM of lactoferrin.   The cell fixing method according to claim 8, wherein the concentration of lactoferrin in the cell solution reduced in concentration by a culture solution not containing lactoferrin is less than 10 μM.   A cell solution containing floating adherent animal cells is placed in the planned fixing portion, and then a revoking substance that invalidates the floating effect of the adherent animal cells is added to the cell fluid in the planned fixing portion, and A cell fixing method for fixing adherent animal cells, wherein the cell fluid contains 5 to 20% of serum and 10 to 500 μM of lactoferrin.   The cell fixing method according to claim 10, wherein the lapsed substance is glycosaminoglycan.   12. The cell fixing method according to claim 11, wherein the concentration of glycosaminoglycan in the cell fluid when the adherent animal cells in a floating state are fixed is 10 to 500 μg / ml.   A cell fixing solution comprising a cell culture solution containing glycosaminoglycan.   A fixing material for fixing the adherent animal cells is coated on the planned fixing portion, and then a cell solution containing the floating adhering animal cells is placed in the fixed fixing portion, and the adhesive animal cells are fixed on the planned fixing portion. A cell fixing method, wherein the fixing substance is an extracellular matrix protein, and the cell fluid contains at least 5 to 20% serum and 10 to 500 μM lactoferrin.   The cell colonization method according to claim 14, wherein the extracellular matrix protein is laminin.