JPH0560950B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application) The present invention provides a surface-contacting biological cell or tissue that exhibits increased affinity and reduced defensive response, as well as imparting to them cell growth specificity. It relates to artificial elements such as cell culture containers and artificial organ equipment, and is used in technical fields spanning cell engineering, tissue culture, medicine, and artificial organ science.

(Conventional technology) Advances in cell culture technology, which controls the growth, differentiation, and development of various living cells such as animal cells, plant cells, bacterial cells, and fungal cells, in an artificial environment have been made in two different ways. It relies on technological improvements: improvements in the culture vessels that come into direct contact with the cells, and improvements in the culture medium that provides nutrients to the cells. Of these, the latter has been the mainstream approach in the past.

JP-A No. 60-18174 discloses that a porous material such as a ceramic sintered body is filled into a bone defect as a prosthetic material, and the porous material acts as a biofilter against collagen fibers and bone destruction cells. A method for inducing new bone formation has been proposed. However, this method passively induces new bone formation by blocking the invasion of bone growth-inhibiting substances, and actively induces new bone formation by improving the surface shape of objects that come into direct contact with living cells or tissues. It does not have the effect of increasing or suppressing the growth rate or controlling the direction of growth.

On the other hand, Japanese Unexamined Patent Publication No. 176339/1984 proposes a blade-type intraosseous implant in which the intraosseous implant portion has a stepped cross structure that communicates with the implant through a large number of holes. This proposal aims to increase the mechanical bonding force to prevent the implant from falling off and wobbling, and similarly to the above, it has the effect of increasing or inhibiting the growth rate of living cells and living tissues, and controlling the growth direction. It is not intended to be demonstrated.

Conventionally, medical devices such as artificial organs that are intended to be implanted in living organisms for long periods of time have been designed to improve their reactivity and affinity with connective tissue cells and reduce the so-called defensive response. Major efforts have been focused on improving the

However, the culture vessels and artificial organs currently in use vary greatly in their macroscopic shapes (plate-like, dish-like, cylindrical, etc.) and their materials, but the types of containers and equipment used vary greatly. The shape of the part of the surface that comes into direct contact with cells and is directly involved in cell proliferation, etc., is left without much thought, and most of the parts are treated with a smooth surface, or
The original flat surface shape of the material remains, and no improvement or research focusing on the fine structure of the surface has been proposed yet.

Furthermore, conventional techniques for controlling the growth direction of biological cells and tissues, for example, techniques for directing the growth of neurites, involve treating cells with chemicals such as fibronectin, laminin, collagen, polyornithine, and NGF (nerve growth factor). There was a method of oriented growth by growing along or toward the site where they exist. in this way,
These chemical substances must be arranged by giving direction to each molecule, and therefore not only do they require extremely sophisticated techniques, but these methods also require
There was a problem with stability, as properties were lost as the chemical substance was inactivated.

In addition, as a conventional method, inducing growth in the direction of the electric field using an electric field in biological tissues and cells is known, but this method has problems such as the influence of the electric field on biological tissues is not fully understood. It was hot.

(Problems to be Solved by the Invention) The present inventors have clarified that the fine structure on the surface of an artificial element, which had been neglected in the past, imparts unique properties and behaviors to living tissues and cells that come into contact with it. The present invention has been completed by successfully solving the various problems mentioned above.

A first object of the present invention is to provide an artificial element, such as a culture container for cells and tissues, a medical device, etc., which has a surface that exhibits increased affinity and decreased biological defense response to living cells and tissues that come into contact with it. is to provide.

Another object of the invention is to obtain an artificial element that makes it possible to control cell proliferation, cell growth, and even nerve regeneration.

Still another object of the present invention is to provide inexpensively and easily an artificial element that has the above-mentioned excellent properties with a long-lasting effect and safety.

(Means for Solving the Problems) The above-mentioned object is to provide a surface having a large number of fine undulations, connective tissue, nerve cells, glial cells, Schwan cells, skin cells, and muscle cells in contact with the surface. This is achieved by an artificial element characterized by exhibiting cell growth specificity for living cells or tissues selected from the group consisting of kidney cells and liver cells.

That is, according to the present invention, in artificial elements such as cell culture containers or medical equipment, cells selected from the group consisting of connective tissue, nerve cells, glial cells, Schwan cells, skin cells, muscle cells, kidney cells, and liver cells can be used. Increasing adhesion and selectivity to cells and tissues and controlling cell proliferation by carving fine undulations mechanically or chemically on the surface that comes into contact with living cells or tissues. Furthermore, if these undulations are formed into grooves, cells can grow and form cell groups oriented along such grooves.

(effect) The structure of the present invention will be explained in detail below along with its operation.

The surface portion of the artificial device of the present invention that comes into contact with living cells or tissues has a rough surface with a large number of fine undulations, or more preferably has a large number of grooves. For example, the width and depth of such grooves are approximately
They are preferably in the range of 0.1 μm to 1000 μm, and do not necessarily need to be parallel to each other, nor do they need to be uniform in width and depth or have a regular shape. That is, depending on the material and shape of the artificial element, the depth and width of the fine grooves can be changed as appropriate within the above range. The cross-sectional shape of the grooves can also be arbitrarily selected such as V-shape, U-shape, dovetail shape, etc. Furthermore, the grooves may have any planar shape, such as straight lines, curves, or waves, and even when they overlap each other to form a complex microscopic surface structure, the above-mentioned specific effect can be achieved.

However, regarding the shape and arrangement of the above-mentioned grooves, it is most preferable to arrange straight grooves having widths and depths within the above-mentioned ranges in parallel with each other. It has been confirmed that this method not only makes it possible to control growth and even nerve regeneration, but also allows living tissues and cells to grow easily, reliably, and in a desired direction with good adaptability. That is, connective tissue cells have a remarkable tendency to grow in the grooves, whereas nerve cells and the like grow on the ridges, and oriented growth along the grooves is achieved.

Furthermore, the above-mentioned properties include biologically active substances, preferably collagen, poly-L-lysine, poly-L-ornithine, laminin, fibronectin, tick plasma, LB factor (artificial lipid membrane) and NGF ( Further enhancement can be achieved by depositing a substance selected from the group consisting of (nerve growth factor).

The material of the artificial element in which the above-mentioned fine undulations are carved is not particularly limited, but usually includes quartz glass; hard glass; soft glass; organic polymer materials such as plastics such as polystyrene and polyvinyl chloride, collagen, cellulose, agar, Metals; Ceramics, such as SiN, BN, apatite, etc.; Silicone rubber; Semiconductors, such as Si, Ge, GeAs, InP,
It is at least one selected from GaSe, InSe, etc., and also includes the surfaces of biopolymers such as collagen plates and plasma clots.

The overall macroscopic form of the artificial element of the present invention, such as a container, a device, etc., is not particularly defined. In other words, the purpose is plate-shaped, plate-shaped, spherical, fibrous, cylindrical, particle-shaped, etc.
Although it can be formed arbitrarily depending on the application, it is important that the surface has the above-mentioned fine undulations at least in the portion that comes into contact with living cells or tissues. In general, artificial elements suitable for medical equipment such as cell culture vessels or artificial organs are flat plates, circular dishes, and have a thickness of 10 μm to 10 cm.
Cylindrical or fibrous spheres with a diameter of 100 μm to 10 cm,
Alternatively, it may have a hollow cylindrical shape with an outer diameter of 10 μm to 10 cm.

According to the present invention, the surface with such fine undulations has unique effects such as cell proliferation, cell adhesion, and cell arrangement control, which were not found on conventional unprocessed surfaces. Demonstrate.
These effects vary depending on the material of the element, and also vary to a certain degree depending on the macroscopic shape of the element. Furthermore, it varies depending on the type of cell or tissue that interacts with the element, the type of animal from which it is derived, sex, age, etc. However, the unique properties caused by the fine undulation structure on the surface, which is a function of the device of the present invention, are universal and remarkable beyond the influence of the above factors, and it is difficult to fully achieve the purpose of the present invention. I can do it.

FIG. 1 is an enlarged perspective view schematically showing the growth of cells on a quartz glass plate subjected to microgroove processing according to the present invention.

In the figure, a plurality of linear fine grooves 2 with a U-shaped cross section are carved in parallel to each other on a quartz glass plate 1,
When living cells are brought into contact with the surface and cultured, neuronal cells NC regenerate neurites AX along the ridges on the surface of the ridges 3 between the fine grooves, but connective tissue cells such as glial cells GC A protrusion is grown in the groove 2 along the groove.

FIG. 2 similarly illustrates the growth of cells C along fine grooves 2 carved on a glass flat plate 1. Furthermore, FIG. 3 shows a state in which cells C grow along fine grooves 2 carved on the surface of plastic fibers 1' parallel to the fiber axis.

In order to form fine undulations on the artificial element of the present invention, a photoresist method, a replica method, a scratch method, a press method, an etching method, etc. can be appropriately applied. For example, lithography technology using photoresist is often applied to processing the surfaces of glass dishes, glass plates, etc. as shown in Figure 2, and lithography techniques using photoresists are often applied to processing the surfaces of glass dishes, glass plates, etc. as shown in Figure 3. A similar method can be applied to micromachining the inner surface of the tube. Additionally, three main methods are used to process plastic dishes, fibers, etc. That is, (1) the above-mentioned lithography method;
(2) a method of taking a replica of a glass plate processed by lithography; and (3) a method of creating scratch grooves on the surface using cut surfaces of microscopic particles or fibers. Furthermore, microfabrication of the surfaces of silicone rubber, plastic tubes, etc. or collagen-based fibers, plates, tubes, etc. may be carried out by a method of making a replica of a glass tube, or by a scratch method.

Figure 4 explains the outline of a method for transferring a fine structure onto plastic, silicone rubber, etc. using the replica method. A replica 5 of silicone rubber or the like transferred onto the material 1 is put to practical use as an artificial element of the present invention.

Figure 5 shows an example of a method for carving fine grooves on the surface of a fibrous material. A metal or glass cylinder with fine grooves carved in the axial direction on the inner wall is used as a prototype 4'. By passing the fibers through and pulling them out in the direction of the arrow, an artificial element of the present invention consisting of fibers 1' having fine grooves 2 carved on the surface can be obtained.

FIG. 6 shows a typical embodiment of the scratch method, in which a comb-tooth or serration-shaped master model 4'' made of a hard material is made of a plastic material with a smaller hardness.
Pressed against the surface of a plate 1 made of rubber or various other materials,
The prototype 4'' and the plate 1 are caused to move relative to each other in the direction of the arrow,
By creating scratches on the board surface,
Carve fine grooves.

(Example) Next, the present invention will be further explained with reference to examples.

Example 1 Spinal dorsal root ganglion cells from adult mice were collected and treated with enzymes such as trypsin and collagenase to isolate the cells, and then fine grooves with a width of 0.5 μm and a depth of 0.2 μm were carved. Cultures were carried out on quartz glass and on a smooth surface of quartz glass without fine grooves. The culture solution was a 1:1 mixture of Hams F-12 culture solution and Dulbecco MEM solution to which progesterone, insulin, and transferrin were added as appropriate. Culture is 5
The experiments were carried out under serum-free conditions for 24-48 hours at 37°C in air containing % carbon dioxide. As a result of the culture, neurites regenerated on the glass plate are shown in FIGS. 7 and 8.

When neurites are regenerated on quartz glass without micro-grooves, as shown in FIG. 8, axon regeneration occurs, but its direction is not constant. On the other hand, on silica glass with fine grooves carved (the fine grooves are not observed because they are below the resolution limit of visible light, with a depth of 0.2 μm and a width of 0.5 μm), the fine grooves are as shown in Figure 7. Axonal regeneration oriented along the direction of the sulcus is observed. In this case, some protrusions may extend beyond the groove, but their length is 20% of the protrusion in the groove direction.
Stay below.

Using elements made of materials such as soda glass, plastic, collagen, and apatite with microscopic grooves, the types of nerve cells, types of animals, ages, and types of perineural tissue were changed and cultured in the same manner as above. As a result, the same results as above were observed, although some differences were observed.

Example 2 Dorsal root ganglion cells and motor neurons in the spinal cord of a chicken fetus 15 days after fertilization were separately taken out.
Cultures were simultaneously grown on both ends of the grooves of a plastic plate with grooves of 5 μm wide and 2 μm deep. As a result, dorsal root ganglion cells and motor neurons grew along the sulcus and formed synaptic connections between them with high efficiency. At this time, the electrical stimulation applied to spinal dorsal root ganglion cells is also detected in motor neurons,
Threshold characteristics specific to synaptic connections were obtained.
(Promoting regeneration of nerve fibers and cells) Example 3 Among the elements of the present invention, the material is glass or plastic, and the surface is 2-10 μm wide and 0.5 μm deep.
Adult mouse spinal dorsal root ganglion cells were cultured using the method of Example 1 on a -1 μm groove. However, in this example, FCS (fetal bovine serum) was added to the medium.
and culture for 84 hours. In addition to neurons, the spinal dorsal root ganglion contains Schwan cells and glial cells.
These cells proliferate during culture. On the device of the present invention, more than 90% of nerve cells grow on the grooves, and conversely, more than 90% of cells other than nerves grow under the grooves. Because of this difference in properties, nerve cells and other cells can be easily separated, and each type can be collected separately. (Function of cell sorter) Example 4 When fibroblasts, Schwan cells, skin cells, skeletal muscle cells, kidney cells, and liver cells obtained from a chicken on the 15th day after fertilization are cultured on the device of the present invention, unique cells are detected. It was found that there is a unique relationship between the device shape, cell selectivity, and cell growth stimulation. In particular, a remarkable effect was observed in culturing cell tissues derived from organs with characteristic cell arrangement. These characteristics were also greatly influenced by the type and age of the animal from which the cells were derived, and the culture method. The optimal element material, groove shape, width, depth, etc. were assigned to each cell. (Selectivity for different types of cells) Example 5 Fine grooves with a width of 10 μm and a depth of 3 μm were carved in a predetermined range on a quartz glass plate, and the area outside the range was left as a smooth surface. When cells were cultured thereon in the same manner as in Example 1, as shown in FIG. 9, in the grooved area 6, growth of nerve cells and connective tissue occurred along the grooves. However, in the portion 7 where the grooves were not processed, the growth was not constant.

Example 6 Using the quartz glass plate used in Example 5, a single type of cell (connective tissue cell) not containing nerve cells was cultured in the same manner as in Example 5. As shown in Figure 10, the grooved part 6
There was a significant difference in the orientation of cell growth between the area 7 and the untreated area 7, and there were also differences in cell adhesion, cell selectivity, etc.

(Effects of the Invention) The present invention provides a cell culture container or a medical device in which biological tissue or tissue selected from the group consisting of connective tissue, nerve cells, glial cells, Schwan cells, skin cells, muscle cells, kidney cells, and liver cells is used. By mechanically or chemically carving fine undulations on the surface that comes into contact with living cells, we can increase adhesion and selectivity to cells and tissues, control cell proliferation, and further improve this undulation. By using striped grooves, especially many linear striped grooves that are parallel to each other, cells and tissues can be oriented and grown along the grooves. It can be done.

That is, the effects of the device of the present invention can be summarized as follows.

1. Cell growth promoting effect A. Effect of promoting cell division and accelerating cell proliferation.

When cells are cultured on this device, the time required for cell division is shortened, and the number of cells that proliferate within a unit of time (for example, one day) increases by at least two times. At this time, various phenomena associated with cell division, such as increases in nuclear DNA and protein synthesis, are simultaneously observed. Such effects are remarkable in cells with excellent cell division ability, ie, liver cells, kidney cells, skin cells, and angiogenic cells.

Effect on growing B cells.

For cells that do not have the ability to divide or have little ability to divide, we can increase the size of the cells,
Alternatively, the effect of lengthening the fibers stretched by cells has been observed. In particular, the fibers of nerve cells increase in length by 2 to 5 times, and the length of skeletal muscle fibers increases by 2 to 3 times and the diameter by 2 to 4 times.

2 Effect of promoting differentiation of cell functions In the living body, cells not only play their roles independently, but also cooperate with surrounding cells and perform their roles while sharing functions. It has been confirmed that the device according to the present invention can exhibit such advanced functions that have not been observed in conventional devices.

A When liver cells and kidney cells are cultured on the device of the present invention, not only the number of cells increases, but also the expression of advanced functions of the cells is promoted.

That is, in liver cells, the activity of enzymes directly involved in detoxification, such as beta-glucuronidase, increases by more than five times. Such an increase in special enzyme activity has not been achieved in conventional culture environments.

Furthermore, in kidney cells, increased activity of multiple enzymes directly related to the cellular urine production and excretion functions was observed.

Furthermore, regarding skin cells, there is an increase in the synthesis of keratin proteins, which is thought to be due to the arrangement of multiple cells, and promotion of synthesis of basement membrane component proteins, etc.
Various phenomena are observed that are thought to promote the formation of a more differentiated skin structure.

B Even in muscle cells and nerve cells that rarely undergo cell division, functional differentiation is promoted to express advanced functions.

Regarding nerve cells, it not only directs the growth of nerve fibers as already mentioned, but also
Special functions of nerves, ie, functions associated with the formation of neural networks, are expressed. That is, the activity of enzymes used by nerve cells to synthesize chemical transmitters, such as choline acetylase and catecholamine synthase, increases. Also, the release of chemical mediators in the culture medium is increased several times. Furthermore, an increase in various enzymes was observed within the nerve cells, which indicates that the nerve function has become more advanced.

For muscle cells, not only their length and diameter increase, but also the activity of creatine kinase, which indicates increased muscle contractile function, increases.

In summary, (1) phenomena that are hardly observed when using conventional cell culture methods are observed when using the device of the present invention, and (2) such phenomena are due to highly differentiated cell functions. This is considered to indicate that the state is approaching the state in which it works in the living body.

The device of the present invention does not involve conventionally known methods such as simply coating the surfaces of culture vessels, medical equipment, etc. with growth-inducing chemicals or processing them using heat or electric discharge. Since the undulations, especially the groove structure, are mechanically or chemically processed, the processing technology is relatively simple and easy, and not only is mass production possible, but it also maintains stable action and characteristics for a long period of time. can do.

In addition, as a conventional method, inducing growth in the direction of the electric field using an electric field in biological tissues and cells is known, but this method has problems such as the influence of the electric field on biological tissues is not fully understood. It was hot. The device of the present invention can be used as a device that helps clarify these issues, and its characteristics can also be used as a cell sorter.

In particular, the device of the present invention is particularly excellent in its cell selectivity and cell arrangement controllability, and can be used as an element for medical equipment to be placed in an organ or an organ that requires a specific cell arrangement. That is, conventionally, for medical devices intended to be implanted in a living body for a long period of time, efforts have been focused on improving the material and shape of the device with the aim of lowering the so-called defensive response of the living body. However, the device of the present invention uses a surface processing method to give unique cell groups a unique harmony, and also has unique cell selectivity that controls the arrangement of cells on the device surface. It can be used as a surface coating element for various medical equipment. It has particularly high reactivity and affinity with connective tissue cells,
Depending on the material of the element and the groove processing, it can be used as a medical device with low biological defense response.

As explained above, the present invention does not have the problems that can occur when using chemical substances or electric fields.
There is an advantage that biological tissues and cells can be easily and reliably grown in a desired direction. Therefore, the present invention can be applied to biotechnology such as specific synapse formation, medical treatment such as promotion of injury healing, and material production, such as providing a new method for extracting materials from cells.

What I would like to particularly emphasize is that the present invention is not limited to the culture vessel material, size, shape, etc., and can change the macroscopic shape without impairing the conventional improvements in material quality and shape. Rather, it is used as an additional technology on top of conventional technology. It is believed that the fine surface processing of the present invention will add previously unseen properties such as cell selectivity and cell proliferation control to existing culture vessels and medical equipment, further improving their performance. . Furthermore, the device of the present invention is not limited by its original material, and can be applied to containers and equipment made of one or more types of materials. It is a ground-breaking invention that has a high possibility of being used as a device, and is expected to have a promising future in the following applications.

1. Artificial organs, medical devices placed in living bodies. Especially as surface elements for artificial blood vessels, artificial hearts, and pacemakers. Also used as materials and elements for nerve sutures and transplants.

2 Biochip, biocomputer.

3 Cell separation equipment (cell sorter), cell cloning equipment 4 Equipment for organ transplants, brain and nerve transplants

[Brief explanation of the drawing]

Figures 1 to 3 are an enlarged perspective view, a plan view, and a perspective view, respectively, illustrating the state in which living cells grow on the artificial device of the present invention, and Figure 4 is a method for manufacturing the artificial device of the present invention. 5 and 6 are perspective views illustrating a method of manufacturing another embodiment of the prosthetic device of the present invention, and FIG. Figure 8, sketched from a microscopic photograph showing the state of regeneration.
The figure is a drawing taken from a microscopic photograph showing the state in which neurites regenerate on a conventionally known artificial element, and Figures 9 and 10 are taken from a microscope photograph to show the effects of the artificial element of the present invention. . 1...Glass plate, 1'...Plastic fiber, 2...
Fine grooves, 3...ridges, 4, 4', 4''...original model, 5...replica.

Claims (1)

[Scope of Claims] 1. A cell comprising a surface carved with a large number of minute undulations, and comprising connective tissue, nerve cells, glial cells, Schwan cells, skin cells, muscle cells, kidney cells, and liver cells in contact with the surface. An artificial element characterized by exhibiting cell growth specificity for living cells or living tissues selected from the group. 2 Claim 1 in which the fine undulations are striped grooves
Artificial elements as described in section. 3 The narrow grooves are approximately 0.1 to 1000 μm wide and approximately 0.1 to 1000 μm deep.
Artificial element according to claim 2, having dimensions of 1000 μm. 4. The artificial element according to claim 3, wherein the grooves are parallel to each other. 5. The artificial element according to any one of the claims above, further comprising a biologically active substance coated on the finely undulated surface. 6. Claim 5 in which the biologically active substance is selected from the group consisting of collagen, poly-L-lysine, poly-L-ornithine, laminin, fibronectin, tick plasma, artificial lipid membrane (LB membrane, etc.), and nerve growth factor. Artificial elements as described in section. 7 The artificial element is quartz glass, hard glass, soft glass, organic polymer material, metal, semiconductors,
An artificial element according to any one of the claims, comprising at least one substance selected from the group consisting of silicone rubber and semiconductors.