Patent Application
20250027037
Present invention relates to a non-woven mat used as a cell culture substrate for growing mesenchymal stem cells (MSCs), and a method for producing the cell culture substrate.
Cell culture is a process of taking cells out of a living organism and keeping them alive. In order to culture cells in vitro, it is necessary to create an environment in vitro that is similar to the microenvironment in vivo.
Recently, non-woven fabrics made of nanofibers produced by using electrospinning method are proposed as a cell culture substrate. Non-woven fabrics made of fibers produced by electrospinning method form a three-dimensional structure that is similar to the extracellular matrix (ECM) and can be used as an excellent three-dimensional cell culture substrate in place of conventional two-dimensional cell culture plates.
Non-woven fabrics used as three-dimensional cell culture substrates must have spaces between fibers where cells can enter and nutrients and oxygen contained in the culture medium can be supplied therein. However, fibers produced by electrospinning method are generally extremely thin, with fiber diameters ranging from several tens of nm to several μm, so the fibers are densely entangled with each other and do not form sufficient gaps between fibers. Therefore, measures have been proposed to secure gaps between fibers (Enhancing cell infiltration of electrospun fibrous scaffolds in tissue regeneration, Jinglei Wu et al. University of TEXAS Bioactive Materials 1 (2016) 56-64).
In order to attach MSCs, which are adhesive cells, to a scaffold, the surface of the scaffold must be covered with adhesive proteins so that cells can attach to the substrate having a protein layer on its surface. Therefore, in order to use non-woven fabrics as cell culture substrates, it is important that surfaces of the fibers that form the non-woven fabrics effectively adsorb the adhesive proteins contained in the culture medium.
It has also been reported that cells can be attached to a scaffold with an uneven surface shape more easily than to those with a planar surface. In this case, if the unevenness is extremely fine, cells cannot recognize the unevenness and it is no different from a planar surface. On the other hand, if the unevenness is too high, cells cannot attach to the scaffold beyond the unevenness.
Furthermore, it has been reported that when a non-woven fabric is used as a cell culture substrate, cells tend to attach at intersections of the fibers and migrate along the fiber alignment from there (Mechanical tensile strengths and cell proliferative Mechanical tensile strengths and cell proliferative activities of electrospun poly(lactic-co-Glycolic acid) composites containing β-tricalcium phosphate Phosphorous Research Bulletin Vol. 26 Special Issue (2012) pp 109-112 Shingo Ito et al).
Non-patent literature 1: Enhancing cell infiltration of electrospun fibrous scaffolds in tissue regeneration, Jinglei Wu et al. University of TEXAS Bioactive Materials 1 (2016) 56-64
In order for cells to proliferate and differentiate in vitro, the cell culture substrate must provide the same environment for cells as in vivo. However, there are a wide range of requirements for cell culture substrates. For this reason, although use of a non-woven fabric as a cell culture substrate has been proposed, its development has not been easy in practice. As a result, cell culture substrates using non-woven fabrics have not been successfully commercialized.
In order to solve above problem, inventors of the present invention made an intensive study and reached the idea of utilizing ReBOSSIS (registered trademark), which is a cotton-like artificial bone previously developed and commercialized by the applicant, for a cell culture substrate. The above cotton-like artificial bone is a bone regeneration material for implantation in a bone defect and has received a high reputation in the market with many clinical results. The cotton-like structure is composed of numerous fibers with diameters of several tens of micrometers, and the spaces between the fibers form the microenvironment necessary for entry, proliferation, and differentiation of osteoblastic cells. In addition, ReBOSSIS contains a large amount of calcium phosphate particles in the fibers by using a kneading method, and the large amount of particles are partially exposed on the surface of the fibers to form a concave-convex shape that is suitable for cell attachment. Inventors reached utilizing this unique structure of ReBOSSIS for a three-dimensional cell culture substrate in vitro for MSC.
Based on the above idea, inventors made a further study and succeeded to modify the production process of ReBOSSIS such that spinning solution emitted from the nozzle are distributed and deposited on the rotating drum collector as many short fibers to produce a non-woven mat with a three-dimensional structure composed of biocompatible fibers. By conducting a culturing experiment by seeding MSCs onto the non-woven mats thus prepared, it was found that the seeded MSCs are effectively trapped by the non-woven mats, attached to the fibers, and shows a high proliferation.
Based on above findings, inventors of present invention have reached a cell culture substrate comprising a non-woven mat made of biocompatible fibers produced by using an electrospinning method,
Further, inventors of present invention have reached a method for manufacturing a cell culture substrate comprising a non-woven mat made of biocompatible fibers produced by electrospinning method, the method comprising:
Preferably, the fibers of the non-woven mat contain 20-30 vol % (about 45-55 wt %) of inorganic filler particles, the fibers are 20 mm-80 mm in length, and a mat shape is formed by the plurality of curved fibers entangled in random directions and adhered each other.
Preferably, the fibers constituting the non-woven mat contain 40-50 vol % (about 65-75 wt %) inorganic filler particles, the fibers are 2 mm-10 mm in length, and the mat shape is formed by a plurality of short curved fibers entangled in random directions and adhered to each other.
Preferably, the inorganic filler particles are HAp particles, and more preferably needle-shaped HAp particles.
Preferably, particle size of the inorganic filler particles is 1 to 5 μm.
Preferably, the non-woven mat is cut to fit the dimension of the cell culture well plate or dish.
Preferably, the non-woven mat has a thickness of 0.1 mm to 0.5 mm.
Preferably, the outer diameter of the biocompatible fibers that form the non-woven mat is 10 to 80 μm, more preferably 10 to 60 μm.
Preferably, the resin fibers comprise PLGA, PLA or PCL.
Preferably, the fibers comprising the non-woven mat are three-dimensionally entangled with a number of fibers with an inter-fiber distance of 10-200 μm, so that mesenchymal stem cells of approximately 10 μm in diameter can penetrate into the spaces between the fibers and be retained therein.
The non-woven mats produced by an embodiment of the method of the invention have an outer diameter of 10 to 80 μm (see
The non-woven mat produced by the method of an embodiment of the invention have numerous filler particles exposed on the surface of the fibers, forming an uneven structure that allows cells to effectively attach to the surface of the fibers.
Fibers constituting non-woven mats produced by the method of one embodiment of the invention have excellent protein adsorption performance because the surface charged inorganic filler particles are exposed without being covered by a resin layer, and cells can adhere to the fiber surface via the proteins adsorbed on the surface of the fibers.
The non-woven mat produced by the method of an embodiment of the invention are formed by short fibers that are torn apart to become a number of short fibers having a length of 20 mm to 80 mm during the flight in the ES apparatus and depositing on the rotating drum collector. The fibers are aligned in a certain direction (see
The non-woven mat produced by the method of an embodiment of the invention is formed by a process in which a number of short fibers of 2-30 mm in length that are torn apart during the flight in the ES apparatus is deposited on the rotating drum collector such that a number of curved short fibers are deposited in random directions to form a mesh structure (see
The non-woven mat produced by the method of an embodiment of the present invention is flexible in three-dimensional directions and is not broken when bending pressure is applied to the non-woven mat, and thus handling of the mat is easy.
The non-woven mat produced by the method of an embodiment of the present invention is made of 2 mm to 80 mm curved short fibers that are deposited and intertwined to form a 0.1 mm to 0.5 mm thickness, forming an excellent three-dimensional culture substrate.
The specific surface area of the fiber constituting the non-woven mat produced using the method of an embodiment of the present invention is significantly increased due to the formation of countless number of bubble pores with diameters of around 1 μm or less throughout the entire surface of the fiber (see
The fibers that form the non-woven mats used as a cell culture substrate of the present invention are biocompatible and biodegradable, allowing the mat having adhesively cultured cells to be transplanted directly into the patient's body.
The non-woven mat used as a cell culture substrate of the present invention can be manufactured inexpensively and efficiently using the electrospinning method similarly to ReBOSSIS (registered trademark), allowing MSCs to proliferate on a commercial basis.
Hereinafter, preferable embodiments of the present invention are explained by referring to the drawings.
As the resin that is fibrillated by using electrospinning method, biocompatible resins such as PLA, PLGA, and PCL can be used as long as they can be dissolved by a solvent. Because biocompatible resin such as PLA, PLGA, and PCL have hydrophobic group, those resins are suitable for the adsorption of hydrophobic portions of proteins. Because PLGA is an amorphous resin, it is suitable for the preparation of a composite by using a kneading process.
When PLGA is used, molecular weight of the resin should be between 150,000 and 400,000 to produce a fiber from the resin using electrospinning method. More preferably, 200,000 to 400,000 is preferred. If the molecular weight is lower than 150,000, entanglement of molecular chains becomes weak, and the resin may not maintain its shape as a fiber. Conversely, if the molecular weight is greater than 400,000, viscosity of the spinning solution becomes too high, and as a result, it becomes necessary to decrease the resin concentration by increasing the proportion of solvent in the spinning solution to lower the viscosity. In that event, amount of solvent in the spinning solution becomes too large. As a result, it becomes difficult to sufficiently volatilize the spinning solution during flight after the spinning solution is ejected from the nozzle, resulting in a difficulty for the spinning solution to become fibers.
Inorganic filler particles used in the present invention are sized such that the particles can be uniformly dispersed in the ES spinning solution and form an uneven structure on the surface of the fiber where the particles are exposed so that cells can easily attach to the fiber. In a preferred embodiment of the invention, particles of 1 μm to 5 μm size are used.
ReBOSSIS (registered trademark) uses bioabsorbable β-TCP particles as a filler because it is implanted in the body. However, bioabsorbability is not necessary for a cell culture substrate that is used in vitro. HAp particles are not bioabsorbable but have positively charged crystal face and negatively charged crystal face in a neutral PH environment. The HAp particles have both positively and negatively charged crystal faces in a neutral PH environment, which gives them excellent protein adsorption performance. Needle-shaped HAp particles are particularly desirable because they have a positively charged a-face in the longitudinal direction of the needle and can effectively adsorb proteins having a negative effective surface charge (adhesive proteins) in a neutral PH environment.
The solvent that is used for the method of present invention is preferably a material that can dissolve a solvent soluble resin, and is highly volatile with a boiling point below 200° C. under atmospheric pressure conditions and are liquid at room temperature. Chloroform is preferred as a volatile solvent for use in the method of the present invention because it has excellent solubility of biocompatible resins and high volatility.
Housing 10 can be shut off from the outside air by closing the front door 11, which is mounted so that the front opening can be opened and closed and the temperature and humidity inside the housing can be adjusted according to individual spinning conditions. The housing 10 is equipped with an exhaust fan to allow ventilation while the equipment is in operation. Rail 31 is mounted near the ceiling of the housing 10, which is a facility for sliding the nozzle 30 in the horizontal direction. In the method of the present invention, temperature and humidity in the housing should be adjusted to 15° C. to 30° C. and 50% or less in order for the spinning solution emitted from the nozzle to deposit on the rotating drum in the form of fibers, since the solvent need to evaporate sufficiently while the emitted fibers are falling and flying inside the housing 10.
Syringe 20 is fixed near the ceiling of housing 10. Syringe 20 may be mounted on rails provided in housing 10, and syringe 20 itself may be configured to slide along with nozzle 30 on the rail.
After the syringe 20 is filled with the spinning solution, the switch to start spinning is pressed, and then the spinning solution filled in the syringe 20 is pushed through the tube to the nozzle 30 at a constant pressure/rate to start the electrospinning process. In one embodiment of the invention, the amount of spinning solution that can be filled into the syringe is set to be 10 ml.
In
Nozzles that can be used in the electrospinning equipment of the present invention are 27 G, 22 G, and 18 G depending on the size of diameter. Sizes of diameter of 27 G, 22 G, and 18 G are shown in the following table.
A DC power supply (PW) having adjustable voltage is connected to nozzle 30. When the DC power supply is turned on, a positive high voltage is applied to nozzle 30, making nozzle 30 a positive electrode. Rotating drum 40, which is electrically grounded, becomes a negative electrode by electrostatic induction, generating an electric field between nozzle 30 and rotating drum 40. At the tip of nozzle 30, positively charged spinning solution discharged from the nozzle is subjected to electrostatic attraction, causing the Taylor cone phenomenon, and is ejected into the air in a form of fibers.
At the bottom of housing 10, rotating drum 40 is installed to collect the fibers produced by electrospinning as a non-woven mat. Rotating drum 30 is electrically grounded. When a positive voltage is applied to nozzle 30, electrostatic induction occurs, and the rotating drum 40 is negatively charged and becomes the opposite electrode of the nozzle 30.
The rotating drum is wound with a conductive aluminum sheet or a highly peelable sheet such as silicone sheet. The fibers emitted from the nozzle 30 and flying down can be wound onto the drum around a rotating take-up shaft to obtain a non-woven mat. At this time, the fibers deposited on the drum are charged by the high voltage of ES, so they repel each other on the surface of the drum where they are deposited. When the drum rotation speed is fast, the fibers have a strong tendency to align and orient in the winding direction, but when the drum rotation speed is slow, the fibers' repulsive force against each other prevails, and as a result, the fibers have a strong tendency to orient in a random direction.
The biocompatible resin and inorganic filler particles are mixed and kneaded in a kneader to produce a composite, and the composite is dissolved in a solvent to obtain a spinning solution. In the present invention, the composition of the biocompatible fibers constituting the non-woven mat is determined by the mixing ratio of the biocompatible resin and inorganic filler particles in the composite prepared by kneader kneading. For example, HAp50 mat is a non-woven mat composed of biocompatible fibers containing 50% by weight (24.2 vol %) of HAp particles and 50% by weight (75.8 vol %) of biocompatible resin. HAp70 mat is a non-woven mat composed of 70% by weight (42.6 vol %) of HAp particles and 30% by weight (57.4 vol %) of biocompatible resin.
In the present invention, a composite with a uniform dispersion of calcium salts in an amount exceeding 20 vol % (about 45 wt %) can be prepared by using the kneading method. A spinning solution having a large amount of inorganic particles uniformly dispersed can be prepared by dissolving the composite thus prepared in a solvent such as chloroform. Details of the kneading method are described in PCT/JP2017/016931 (WO2017/188435). Resin concentration of the spinning solution must be below a certain level to be able to smoothly deliver the spinning solution from the syringe to the nozzle. On the other hand, resin concentration of the spinning solution must be above a certain level in order for the resin to act as a binder for the filler particles to form continuous fibers.
True density of calcium compound particles is higher than that of PLGA. For example, PLGA has a density of 1.01 g/cm3, HAp has a density of 3.17 g/cm3, and β-TCP has a density of 3.14 g/cm3. Therefore, wt % and vol % correlate as follows:
In ReBOSSIS (registered trademark), composition of calcium salts (bone formation factor) in the fibers comprising the cotton shape is controlled by weight percent. However, since in the present invention it is important that the large amount of inorganic filler particles are physically exposed on the surface of the fiber to form an uneven structure to which cells can easily attach, it is reasonable that the amount of inorganic filler particles contained in the fiber is determined by volume that the particles occupy in the fiber, not by weight percentage. Therefore, in the present invention, the amount of inorganic filler particles is indicated by converting weight percentages to volume percentages based on the content correlation table above.
In the method of the present invention, the spinning solution filled in the syringe is delivered at a faster rate than normal ES. As a result, the flow rate per second of the spinning solution at the nozzle outlet becomes larger. In an embodiment of present invention, when the spinning solution filled in the syringe is fed at a rate of 3 ml/h to 15 ml/h to the discharge port having an inner diameter of 0.4 mm to 1.0 mm, flow rate of the spinning solution by volume at the nozzle outlet is 0.83 mm3/sec to 4.2 mm3/sec, mass flow rate is 1.2 mg/sec to 6.8 mg/sec. As the volume per unit time of the spinning solution extruded from the nozzle outlet increases, the force that causes the spinning solution ejected from the nozzle to fall increases under the action of gravity due to its own weight. As a result, the degree of swinging of flight trajectory of the emitted spinning solution is decreased because repulsive force due to the bias of the electric charge becomes smaller compared to the case where the spinning solution is pulled down to the drum collector only due to the pulling force of the electric field.
Upon filling the spinning solution into the syringe, DC power supply is turned on and voltage is applied to the nozzle 30. By applying voltage to the nozzle 30, the filled spinning solution is charged and a potential difference is created between the installed drum collector and the nozzle, causing the charged spinning solution to be pulled toward the drum by the Taylor cone phenomenon.
In the method of present invention, the spinning solution prepared using the kneading method contains a large amount of inorganic filler particles. When the spinning solution is ejected from the nozzle in the form of fibers, the inorganic filler particles are held together with resin as a binder, and form longitudinally continuous fibers in that state. However, if the flight trajectory is violently shaken by the repulsive force caused by the bias of the electric charge during the flight in the ES apparatus, the filler particles becomes unable to maintain the state of being held together by the resin, and the fibers are torn off during the flight process, resulting in formation of a number of short fibers, which are then deposited on the rotating drum collector.
In the method of the present invention, the spinning solution filled in the syringe is fed at a high feed rate to the discharge port of the nozzle having a large diameter, so a large amount of spinning solution is pushed downward from the discharge port per unit time, and the extruded spinning solution falls downward by gravity. At the same time, the solution is pulled toward the collector by the force of the electric field generated between the nozzle and the collector by applying high voltage to the nozzle. The pulling force due to the electric field is subjected to a repulsive force caused by the bias of the electric charge, and the flight trajectory swings. However, reduction of fiber diameter caused by the oscillation of fiber flight path which occur in the bending instability phenomenon of usual electrospinning does not occur. Only reduction of fiber diameter that is caused by evaporation of solvent and swinging of flight trajectory occurs.
In order for the phenomenon of swinging flight trajectory of the present invention to occur, the electric field formed between the nozzle and the rotating drum must be above a certain degree. The strength of the electric field is determined by the value of the applied voltage and the distance between the nozzle 30 and the rotating drum 40 under the formula V=Ed. Therefore, the value of the applied voltage required to generate the swinging of flight trajectory phenomenon cannot be determined alone. However, the value of the applied voltage is necessarily set higher than a certain value because the flight distance of the fiber emitted from the nozzle must be greater than a certain distance because the solvent must be evaporated during that time of flight. In one example of this invention, the distance between the nozzle and the drum is 200 mm, and the voltage applied to the nozzle is set to be 28 kV.
By reciprocating the spinneret horizontally for 1 hour at a moving speed of 2 cm/s with a moving width of 10 cm while spinning fibers from the nozzle, the short fibers are distributed and deposited on the rotating drum collector, and the fibers adhere and bond to each other in that state to form a non-woven mat. Thickness of the non-woven mat should be about 0.1 mm to 0.5 mm in order to seed MSCs over the entire area of the mat while maintaining the three-dimensional structure required for a three-dimensional cell culture substrate, and to detach and collect the cells that have been seeded, attached to the mat, and proliferated from the mat.
After depositing the fibers as a non-woven mat on a rotating drum that is wound with a peelable sheet such as aluminum or silicone sheet, the mat can be collected by removing the aluminum sheet from the rotating drum. In the method of the present invention, multiple short fibers reach the drum in a winding state as they are shaken out of their trajectory during the falling flight. Thus, the multiple short fibers, which are wound and curved, adhere and bond with each other on the drum to form a non-woven mat.
The fiber produced by the method of the present invention has countless number of bubble pores of around 1 μm or smaller diameter throughout the entire surface of the fiber. The mechanism by which such bubble pores are formed is that the fiber emitted from the nozzle contains a large amount of chloroform, and the chloroform contained in the fiber evaporates as bubbles inside and on the surface of the fiber during flight. When the bubbles generated inside of the fiber go out, several bubbles combine to reduce the surface area and become larger bubbles that go out of the fiber. Bubbles generated near the surface of the fiber are smaller than those generated inside the fiber. Fibers emitted from the nozzle are constantly exposed to fresh air as they fly through the air and continue to receive thermal energy, which accelerates vaporization near the surface. By receiving a lot of energy, a large number of bubbles are generated, and it is considered that some of them combine to reduce the surface area, causing the bubbles to become larger.
Size of the bubble pores formed on the surface of fibers spun by this method is determined by the viscosity of the polymer. In an embodiment of the present invention, diameter of the bubble pores formed by the air bubbles breaking out was 0.1 to 3 μm. To form bubble pores on the surface of the resin fibers, it is effective to blow air in the ES apparatus.
The fibers that form the non-woven mat of the present invention have the ability to adsorb adhesive proteins contained in the culture medium on the surface of the fibers.
As for the mechanism by which proteins are adsorbed on the surface of substrates, it is known that proteins are adsorbed on the surface of polymer materials through hydrophobic interactions originating from the protein molecules themselves. Proteins are high polymers consisting of amino acids with hydrophilic and hydrophobic groups, and the surfaces of protein molecules have a mosaic structure consisting of hydrophilic and hydrophobic portions. Resins such as PLA, PLGA, and PCL can be used suitably because they have hydrophobic groups.
Furthermore, amino acids that constitute the protein molecule have both amino groups and carboxyl groups, and thus have an isoelectric point. Acidic amino acids having multiple carboxyl groups have a lower isoelectric point, while basic amino acids having multiple amino groups have a higher isoelectric point. Therefore, in a neutral PH solution, acidic proteins tend to be negatively charged and basic proteins tend to be positively charged. Ceramic particles have high surface energy and their surfaces are positively or negatively charged. Therefore, when ceramic particles are immersed in a neutral PH medium containing proteins, acidic proteins are more likely to be adsorbed on particles with positively charged surfaces because their effective surface charge is negative in a neutral PH environment, and basic proteins are more likely to be adsorbed on particles with negatively charged surfaces because their effective surface charge is positive.
Protein adsorption based on electrostatic interactions is more adsorptive than that based on hydrophobic interaction. Therefore, in order to obtain high initial cell attachment to the scaffold material, the surface of the fiber should be charged opposite to the effective surface charge of the protein. HAp can be suitably used because the Ca2+ present on the particle surface adsorbs acidic proteins having negative effective surface charge under neutral pH.
Fibers produced by using the electrospinning method of the present invention have inorganic filler particles exposed on the fiber surface to form an uneven structure thereon, but it is possible that the exposed particles are covered by a thin resin layer. Since the surface charge of inorganic particles is weak, if the surface of the particles is covered by a resin layer, even if it is a very thin layer, the ability of the particles to adsorb proteins is lost. Based on this consideration, thinking of the fact that HCL dissolves HAp but not resin (PLGA), the inventors of this invention conducted an experiment in which fibers containing HAp particles were immersed in HCL solution and observed whether the fibers were covered by resin by observing whether HAp was dissolved or not.
Experiment 1 in which experimental sample of non-woven mat (HAp50 mat) consisting of fibers produced by electrospinning using a spinning solution prepared by preparing a composite of 50 wt % (24.2 vol %) of HAp particles and 50 wt % (75.8 vol %) of PLGA resin by kneading and dissolving it in chloroform and Experiment 2 in which a sample of non-woven mat (HAp70 mat) consisting of fibers produced by the electrospinning method using a spinning solution prepared by preparing a composite of 70 wt % (42.6 vol %) HAp particles and 30 wt % (57.4 vol %) PLGA resin using the same kneading method, and dissolving the composite in chloroform were conducted.
<HAp50 mat>
<HAP70 mat>
Experiments were conducted to confirm the adsorption of proteins to the mat using serum albumin (BSA) and fibronectin, adhesive protein, as proteins to be adsorbed on the inorganic particles.
Non-woven mat samples formed of PLGA resin fibers produced by electrospinning method: a sample containing 50 wt % HAp and a sample containing 50 wt % β-TCP were prepared. Each sample was immersed in serum-free medium containing adhesion protein (fibronectin). Suspension of MSC (ADSC Lonza) suspensions were seeded. Attachment and proliferation of the cells of each sample was observed and compared.
Each non-woven mat SAMPLES were treated by immersing them in an amphiphilic 70% ethanol and serum-free medium solution so that they would not repel immersion in water and medium, and then used in 24 well plates.
Cell suspension (50,000 cells/500 μl) was seeded dropwise onto each non-woven mat sample in a 24 well plate.
The number of cells that attached and proliferated on each non-woven mat sample was measured by MTT assay and absorbance analysis. The measurement results are shown in
Since the non-woven mat is formed of biocompatible and biodegradable fibers, it can be transplanted into a living body with cells attaching to it. The fact that such high cell attachment and proliferation could be achieved in the present invention while using such cell culture substrates was a groundbreaking achievement in this technical field.
The present invention has been described above in the example of a non-woven mat sheet made of fibers produced by using electrospinning method. However, the sheet is not limited to a non-woven mat as long as it can be used as a cell culture substrate and has a structure that allows cells to penetrate and adhere to it.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/043735 | 11/28/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63284638 | Dec 2021 | US |
