KR20210147970A - nano-cellulose materials for 3d bioprinting, Method for preparing nano-cellulose and applications thereof - Google Patents

Abstract

The present invention relates to a nano-cellulose composition for 3D bioprinting, a method for preparing the same, and an application method thereof. The present invention can provide functional bioparticles for cosmeceuticals based on nano-cellulose using a 3D bioprinting technology. The nano-cellulose composition for 3D bioprinting includes an epidermal growth factor, low viscosity nano-cellulose, and a non-ionic surfactant. According to the present invention, the composition is non-toxicity and biologically safe.

Description

Nanocellulose for 3D bioprinting, manufacturing method of nanocellulose for 3D bioprinting, and application thereof {nano-cellulose materials for 3d bioprinting, Method for preparing nano-cellulose and applications thereof}

The present invention relates to a nanocellulose composition for 3D bioprinting, a method for manufacturing a nanocellulose composition for 3D bioprinting, and utilization thereof.

More specifically, it provides a nanocellulose composition for three-dimensional bioprinting comprising epithelial cell growth factor (EGF), low-viscosity nanocellulose and a nonionic surfactant, a manufacturing method thereof, and utilization thereof.

Recently, safety and harmfulness issues for products closely related to our lives, such as humidifier disinfectants, heavy metal cases in water purifiers, harmful substances in cosmetics, and carcinogens in sanitary napkins, are emerging. are doing

In particular, since microplastics (microbeads), which have been widely used in cosmetic compositions, can cause environmental pollution and cause harm to the human body, in July 2017 in Korea and in the United States in 2018, The sale of cosmetics is also completely banned. This trend is gradually expanding worldwide.

Moreover, in July 2017 in Korea, and in the United States in 2018, not only the manufacture of cosmetics containing microplastics but also the sale of cosmetics were completely banned, and the increasing consumer demand for safer products at home and abroad, The market demand for superior eco-friendly products and materials is increasing.

Each cosmetic-related company is striving to discover materials that can replace synthetic compounds, including microplastics, but their use is limited due to limitations in the supply, functionality, and usability of raw materials, and they have excellent biological safety and functionality. Therefore, interest in the development of functional materials for cosmeceuticals that are safe, functionally excellent and highly useful is increasing.

In order to solve the above-mentioned problems, the present invention is to provide a nanocellulose composition for 3D bioprinting that is non-toxic and biologically safe capable of 3D bioprinting and a manufacturing method thereof.

The present invention relates to a bioparticle based on a nanocellulose composition supported with a functional material capable of uniformly and precisely supporting a functional material such as EGF, a drug, and the like, and a manufacturing method thereof by 3D bioprinting.

The present invention provides a stabilization technique in which activity is maintained for a long period of time compared to the prior art in which a functional material is not supported on low-viscosity nanocellulose, and for this purpose, material loading, cell culture, stem cell culture, organoid and artificial organ culture It can be expanded to a bio-ink material for

However, the problems to be solved by the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

One aspect of the present invention includes epidermal growth factor (EGF), low-viscosity nanocellulose and a nonionic surfactant, wherein the low-viscosity nanocellulose is 1 cp or more and 10 cp or less, three-dimensional bioprinting It provides a nanocellulose composition for use.

According to an embodiment, the cell growth factor has a concentration of 0.005% to 0.015% compared to the total nanocellulose composition, and the nanocellulose is nanocellulose fibrils having a concentration of 1.51% to 1.55% compared to the total nanocellulose composition ( CNF).

According to one embodiment, the nanocellulose composition may include beads having a diameter of 30 μm to 90 μm.

According to one embodiment, the nanocellulose composition may include an epithelial cell growth factor of 10 ppm to 20 ppm per one bead.

According to one embodiment, the nanocellulose may be one in which the hydroxyl group on the surface of the cellulose is surface-modified with a carboxy group.

Another aspect of the present invention, low-viscosity nano-cellulose manufacturing step; mixing the low-viscosity nanocellulose and an epithelial cell growth factor (EGF) solution; and an inkjet nozzle ejection step for 3D bioprinting of the mixed solution; It provides a method for manufacturing a nanocellulose composition for three-dimensional bioprinting, comprising a.

According to one embodiment, the low-viscosity nano-cellulose manufacturing step. preparing a nanocellulose suspension; Nanocrystallization using a grinder or a high-pressure homogenizer; and a step of controlling the viscosity of nanocellulose performed by ultrasonic treatment; may further include.

According to one embodiment, the nanocellulose may be in a concentration of 1.51% to 1.55% of the total nanocellulose composition.

According to an embodiment, the size of the nanocrystallized nanocellulose may be 2.5 nm to 7.0 nm.

According to an embodiment, the low-viscosity nanocellulose may include 1.0 mmol to 2.5 mmol of carboxyl groups per 1 g of nanocellulose.

According to an embodiment, the mixing step of the low-viscosity nanocellulose and the epithelial cell growth factor (EGF) solution comprises a low-viscosity nanocellulose at a concentration of 1.0% to 2.5% and an epithelial cell growth factor solution at a concentration of 0.01% to 0.05%. It may be mixing at a mixing ratio of 2.5 to 1.5: 1.

Another aspect of the present invention provides bio-particles in which a functional material is supported on the nanocellulose composition for three-dimensional bioprinting according to an aspect of the present invention described above.

Another aspect of the present invention, the 3D bioprinting step of filling the nanocellulose composition for 3D bioprinting according to an aspect of the present invention described above in a 3D bioprinter; and three-dimensional printing of a target tissue-like organ; provides an organoid manufacturing method comprising.

Another aspect of the present invention provides an organoid prepared according to the method for preparing an organoid according to an aspect of the present invention.

The present invention prepares low-viscosity nanocellulose from a bio-ink material capable of 3D bioprinting while having biocompatibility, and a functional material (eg, EGF, vitamins, drugs, microorganisms, drugs) using 3D bioprinting technology. transmission material, etc.)

The present invention is a technology for manufacturing nanocellulose with biocompatibility, and lowering the viscosity by setting conditions to enable this, although existing nanocellulose does not perform well in bioprinting. and uniformly EGF-supported nanocellulose-based bio-particles were manufactured.

1 is an exemplary view showing a process of a manufacturing method according to the present invention, according to an embodiment of the present invention.
2 shows the velocity and size of droplets according to Example 1 of the present invention.
Figure 3 shows the bead size according to Example 2 of the present invention.
4 shows the cell stability results according to Example 3 of the present invention.
Figure 5 shows the results of analysis of the EGF loading capacity according to Example 4 of the present invention.
6 shows a low-viscosity nanocellulose according to Example 5 of the present invention.
7 shows a nanocellulose image according to Example 6 of the present invention.
8 shows the analysis of the rheological properties of the aqueous nanocellulose solution according to concentration (dynamic shear viscosity by concentration of the nanocellulose aqueous solution) according to Example 7 of the present invention.
Figure 9 shows the printing suitability evaluation results of the nanocellulose ink according to Example 8 of the present invention.
10 shows the evaluation of the size and monodispersity of micro particles according to inkjet printing parameters according to Example 10 of the present invention.
11 shows the preparation and physical property evaluation of a formulation-controlled monodisperse nanocellulose bioparticle dispersion solution according to Example 11 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of the user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Like reference numerals in each figure indicate like elements.

Throughout the specification, when a member is said to be located "on" another member, this includes not only a case in which a member is in contact with another member but also a case in which another member exists between the two members.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components.

The present invention relates to a nanocellulose composition that contains a natural raw material and a functional material and can be utilized as a material for a three-dimensional bioprinter, and the present invention relates to a nanocellulose composition for a high-quality/high-yield bio-ink. In addition, the present invention relates to a nanocellulose-based high-density uniform material loading and bioparticles using 3D bioprinting technology, and a method for manufacturing the same.

1, the present invention develops nanocellulose for three-dimensional bio-inkjet printing, provides a three-dimensional bioprinting technology capable of supporting EGF in a nanocellulose matrix, and uses the technology to regenerate skin It is possible to provide functional bioparticles for cosmeceuticals in which EGF, which is one of them, is uniformly supported.

The present invention can manufacture nanocellulose for three-dimensional bio-inkjet printing with excellent biological safety (cytotoxicity, skin irritation test, etc.) and have physical properties that can be used as bio-ink, and supports functional material using three-dimensional bio-printing Technology and precise control (size, composition, content) is possible. In addition, it can be utilized for functional cosmeceuticals, and it is possible to provide a manufacturing technology of materials for functional cosmeceuticals.

In the present invention, as a method of controlling the viscosity, it may be possible to adjust the viscosity by other methods such as pH control and temperature control as well as ultrasonic cleaning.

The scope of application of the technology and material according to the present invention can be expanded to organoids, artificial organs, stem cell culture, 3D cell culture, etc. as well as material loading.

The nanocellulose composition for 3D bioprinting according to an embodiment of the present invention includes epithelial cell growth factor (EGF), low-viscosity nanocellulose, and a nonionic surfactant.

The epithelial cell growth factor can improve organic bonding and biocompatibility with human tissue organs in organoids and tissue culture using the composition according to an embodiment of the present invention, and through control of the viscosity of nanocellulose, under certain conditions It can also be easily bioprinted in a desired shape.

Hereinafter, the embodiments of the present invention will be described in more detail, which is intended to help a specific understanding of the present invention, and the scope of the present invention is not limited thereto.

Preparation Example: Establishment of oxidation reaction conditions for manufacturing low-viscosity nanocellulose for 3D bioprinting

<Experiment method>

Cellulose 300 g, NaClO 4 mmol/g cellulose, NaBr 0.02 g/g cellulose, and TEMPO 0.1 g/g cellulose were put in 20 L of distilled water and reacted at 30° C. for 4, 6, and 8 hours, respectively. After changing the NaClO concentration to 8 mmol/g cellulose, the reaction was performed under the same conditions, and each of the six experimental groups was subjected to grinding treatment with a stone spacing of 60 μm - 4 times to prepare a nanocellulose hydrogel.

After storing the prepared nanocellulose in a deep freezer for 24 hours, the frozen experimental group was freeze-dried for 3 days to obtain powder. The degree of surface modification (Carboxylate contents, C.C) was measured by the electric conductivity titration method, and the results are shown in Table 1.

<Comparison of surface modification according to reaction time>

NaClO concentration Surface modification rate (Carboxylate contents, mmol/g cellulose) 4 hours 6 hours 8 hours 4 mmol/g cellulose 1.2 1.4 1.4 8 mmol/g cellulose 1.8 2.0 2.1

When the concentration of NaClO was low (4 mmol/g cellulose), the CC value did not increase significantly even when the reaction time was increased. Shows CC value. In order to secure a C.C value of 1.5 mmol/g cellulose or more, it means that NaClO of at least 4 mmol/g cellulose must be added.

Reaction time (hr) Surface modification rate (Carboxylate contents, mmol/g cellulose) 4 mmol/g cellulose 6 mmol/g cellulose 8 mmol/g cellulose 4 hours 1.0 1.8 1.9

Comparison of surface modification degree according to NaClO concentration

After fixing the reaction time to 4 hours, as a result of performing the reaction with various concentrations of NaClO, it was confirmed that satisfactory carboxylate contents values were obtained at 6 mmol/g cellulose or more.

Example 1: EGF-CNF Bead Inkjet Printing

<Experiment method>

0.03% human EGF solution was mixed with 2.3% CNF in a 1:2 ratio to prepare 1.53% CNF ink containing 0.01% EGF to a final concentration. Printed with an 80-μm diameter inkjet nozzle. Printing conditions including rise time, dwell time, fall time, voltage, and frequency were adjusted to optimize the speed, shape, and size of the ejected droplets. Through the drop analysis function included in the printing system, the speed and size of the ejected droplets were confirmed. The results are shown in FIG. 2 .

<Experiment result>

It was confirmed that 1.53% CNF ink loaded with 0.01% EGF was well jetted as a single droplet.

We searched for printing conditions capable of forming droplets entering within a volume of 100 pL and a diameter of 60 μm.

Example 2: Bead Size Control

<Experiment method>

0.03% human EGF solution was mixed with 2.3% CNF in a 1:2 ratio to prepare 1.53% CNF ink containing 0.01% EGF to a final concentration. _{EGF-CNF was inkjet-printed in a 2% CaCl 2} water bath containing 1% Pluronic F-127 to form beads with a volume of 100 pL. The bead size was confirmed by acquiring a bead picture with an optical microscope. A size of 120 beads per 5 independent samples was adopted. The results are shown in FIG. 3 .

<Experiment result>

Bead sizes for 5 independent samples were measured with an optical microscope, and it was confirmed that beads were formed with a size within 60 μm.

Example 3: Cell Compatibility

<Experiment method>

0.03% human EGF solution was mixed with 2.3% CNF in a 1:2 ratio to prepare 1.53% CNF ink containing 0.01% EGF to a final concentration. _{EGF-CNF was inkjet-printed in a 2% CaCl 2} water bath containing 1% Pluronic F-127 to form beads with a volume of 100 pL. Beads were collected, added to a cell culture multiwell plate, and cultured for 5 days, and cell proliferation and cytotoxicity were checked every other day using the CCK-8 colorimetric assay kit. The results are shown in FIG. 4 .

<Experiment result>

The proliferation rates of human dermal fibroblasts for 5 independent samples were compared. The sample loaded with EGF in the bead state showed a high proliferation rate on day 5. It was predicted that constant EGF release was achieved until the 5th day.

Biocompatibility (biological safety) and printing performance can be confirmed through the official test report. In the cytotoxicity test (ISO 10993-5), negative, (88.9 ± 2.0) % results were confirmed.

In the skin irritation test (primary skin irritation test: skin patch test, IS-MBIQ-001), 2% nanocellulose was subjected to a human skin primary irritation test according to the PCPC Guideline. Reactivity: 0.00) was confirmed.

Example 4: EGF Quantification

<Experiment method>

0.045% human EGF solution was mixed with 2.3% CNF in a 1:2 ratio to prepare 1.53% CNF ink containing 0.015% EGF to a final concentration. _{EGF-CNF was inkjet-printed in a 2% CaCl 2} water bath containing 1% Pluronic F-127 to form beads with a volume of 100 pL.

Five independent samples were prepared by printing 100,000 100 pL droplets per sample. 100,000 printed beads were homogenized with microtube pestle, diluted to a detectable concentration range, and quantitative ELISA assay was performed. As a result of colorimetric assay, the amount of EGF contained in the sample was confirmed. 5 is shown.

<Experiment result>

Human EGF quantification experiments were performed on 5 independent samples. It was confirmed through quantitative ELISA assay that each 100pL volume prepared by inkjet printing contained 15pg (14.996 pg) of EGF. This means that each bead contains more than 15 ppm of EGF.

Example 5: Control of Low Viscosity Nanocellulose

As shown in FIG. 6a, the prepared 3% nanocellulose hydrogel was diluted with distilled water to prepare a nanocellulose dilution of 0.5 and 0.8%, respectively, and then the change in viscosity before and after sonication (Visco QC300, Anton Paar, measurement conditions) : Din adapter applied, repeated 10 times measurement at 25 ℃, 150 rpm) was measured. Ultrasonication (frequency: 20 kHz, 10 minutes) was used to prepare low-viscosity CNF (15cp → 8cp), and in the case of 1% CNF hydrogel, 25cp → 8cp (Fig. 6b).

Ultrasonication method was applied to lower the viscosity of nanocellulose, and as a result of comparing the viscosity before and after treatment, it decreased from 25cp to 8cp, which can satisfy the printable viscosity of 20cp or less.

Example 6: Preparation of Nanocellulose with Uniform Diameter

(1) Experimental conditions

Treatment sample: Cellulose suspension reacted in 6 mmol/g cellulose NaClO for 4 hours

(2) Experimental method

Nanocrystallization using a grinder, and nanocrystallization using a high pressure homogenizer (High Pressure Homogenizer).

① Applied pressure : ~20,000 PSI

② Number of processing: 4 pass

The size and shape of the nanocellulose prepared according to each condition was observed using a TEM (Transmission Electron Microscope).

A comparison between the grinding method and the high-pressure homogenizing method (HPH) is shown in FIG. 7a. The size change of the nanocellulose prepared using the HPH method according to the reaction time is shown in FIG. 7b.

That is, as a result of preparing nanocellulose by another nanofibrillation method, it can be seen that the diameter of CNF prepared using a high-pressure homogenizer was more uniform, and the diameter of CNF decreased as the reaction time increased.

Example 7: Analysis of rheological properties of nanocellulose aqueous solution according to concentration

As shown in FIG. 8 , when the concentration of CNF is high, it is not discharged in the form of droplets because it is higher than the limit viscosity, and in the case of the developed low-viscosity CNF, a printable shear viscosity can be formed even at a high concentration of 1%.

In the case of the initially prepared nanocellulose aqueous solution based on the measured dynamic shear viscosity, it was predicted that inkjet jetting would be possible only at a concentration of 0.5% or less. In order to solve this problem, the possibility of printing was confirmed by preparing sonicated nanocellulose. As can be seen from the red graph, in the case of the new aqueous solution of nanocellulose treated with ultrasonication, it can be confirmed that a printable shear viscosity of less than 20 mPa·s is formed even at a concentration of 1%.

Example 8: Evaluation of printing suitability of nanocellulose ink

Since 3D bio inkjet printers use tens of micro-diameter nozzles to ^{discharge ink in units of picoliters (10 -12} L), it is very important to optimize the printing conditions to find an ink discharge environment with straightness, A straightness test was performed according to various ejection environments such as speed and number of ejection strokes. The results are shown in FIG. 9 .

A straightness test was performed according to various ejection environments such as ejection amount, ejection speed, and number of ejections, and optimal printing conditions for CNF ink exposed as a single droplet were established.

Example 9: Inkjet-bath-based bioparticle manufacturing process development

Cross-linking was induced by forming an ionic bond using a cationic counter ion, and Pluronic F-127 (PE-127) was added so that bioparticle formation proceeds faster and more stably. This provided a role to assist in maintaining the shape of the droplet.

The nanocellulose prepared in this study can be crosslinked by forming ionic bonds using a cationic counter ion because the OH group on the cellulose surface is modified with COO- and a negative charge is added. For this purpose, a non-toxic and eco-friendly process, CaCl ₂ aqueous solution was selected as a water tank, and nanocellulose droplets discharged by inkjet printing meet with the water tank to form an ionic bond to form hydrogel bioparticles.

Addition of Pluronic F-127 (PE-127) to _{CaCl 2} aqueous solution for faster and more stable bioparticle formation. PF-127 is an FDA-approved, biocompatible, nonionic surfactant that assists in maintaining the shape of the nanocellulose droplets until they are cured through ionic bonding when the _{nanocellulose droplets are supported in a CaCl 2 tank.}

By varying the CaCl ₂ concentration and whether or not PF-127 was added, the optimal process conditions for bioparticle formation were established. When CaCl ₂ exists alone, the curing rate is slow at 2-4% CaCl ₂ concentration, and it is confirmed that the curing rate is slow before hydrogelation, and it is cured in a dissolved form rather than a droplet form.

In the case of 8% CaCl ₂ , hardening occurs without dispersing in the water tank, but hardening occurs rapidly from the surface of the water tank before the droplets ejected from the ink jet are completely immersed in the water tank solution, forming bioparticles with a very large diameter and flat shape. confirm that you do. In order to solve this problem _{, PF-127 was additionally added to a low-concentration CaCl 2} aqueous solution to stably maintain bead-shaped droplets until hardening occurred.

Example 10: Microparticle size and monodispersity evaluation according to inkjet printing parameters

As shown in FIG. 10 , the applied voltage was optimized to unipolar 70V, rise time 5us, dwell time 10us, and fall time 5us, and droplets corresponding to a volume of 100pL were discharged.

Example 11: Formulation Controlled Monodisperse Nanocellulose Bioparticle Dispersion Solution Preparation and Physical Property Evaluation

By optimizing the bio-inkjet printing conditions and water tank environment, a dispersion solution of nanocellulose bioparticles having a size in the range of 20 to 60 μm was prepared, and the agglomeration phenomenon was solved by adjusting the gap between the droplets by setting the stage movement path of the inkjet printer. As shown in FIG. 11 , a technology for preparing a nanocellulose bioparticle dispersion solution without bioparticle aggregation and damage was secured, and bioparticles having a uniform size and shape of about 50 μm were prepared.

Example 12: Bioparticle size measurement and uniformity evaluation

The average diameter and uniformity were calculated by randomly extracting 120 bioparticle diameter data measured for each sample, and uniform bioparticles were prepared with an average diameter of about 50 μm and a uniformity of about ±5%.

Example 13: Comparative test for water retention

In this study, a water retention test was performed on bioparticles (1 wt% CNF with EGF) loaded with EGF on nanocellulose prepared under optimal conditions.

The weight of the empty beaker was accurately measured, and 40 g of EGF-supported 1% nanocellulose aqueous solution was placed in 5 100 mL beakers, respectively, to prepare a sample, and as a control, 40 g of tertiary distilled water to which nothing was added was put in a separate beaker and prepared.

After accurately measuring and recording the weight of all samples including the control group, they were placed in a drying oven previously set at 70 ° C. .

<Calculation formula for water evaporation rate>

That is, it showed an evaporation rate of less than 70% compared to the control group, and showed an evaporation rate of 81.67% after 2 hours, which satisfies the target performance index of 90% or less compared to the control group.

The present invention can provide high biocompatibility and biological safety based on the naturally-derived material, and exhibit excellent stability and preservation through high moisturizing power and encapsulation of nanocellulose. 3D bioprinting can be carried out with a high-density material, and reproducibility and uniformity can be secured through 3D bioprinting, for example, as a material for cosmeceuticals, various functionalities can be imparted and it can be used for EGF loading as a functional material.

The nanocellulose material of the present invention has a level of biological safety that can be used as a cosmetic and medical material.

As described above, although the embodiments have been described with reference to the limited embodiments and drawings, various modifications and variations are possible from the above description by those skilled in the art. For example, even if the described techniques are performed in an order different from the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or substituted by other components or equivalents Appropriate results can be achieved. Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (14)

Contains epidermal growth factor (EGF), low-viscosity nanocellulose and a nonionic surfactant, wherein the low-viscosity nanocellulose is 1 cp or more and 10 cp or less, a nanocellulose composition for three-dimensional bioprinting.
According to claim 1,
The epithelial cell growth factor has a concentration of 0.005% to 0.015% compared to the total nanocellulose composition,
The nanocellulose is a nanocellulose fibrils (CNF) having a concentration of 1.51% to 1.55% compared to the total nanocellulose composition, a nanocellulose composition for three-dimensional bioprinting.
According to claim 1,
The nanocellulose composition, comprising beads having a diameter of 30 μm to 90 μm, a nanocellulose composition for three-dimensional bioprinting.
4. The method of claim 3,
The nanocellulose composition is, per one bead 10ppm to 20ppm of the epithelial cell growth factor comprising a, three-dimensional bioprinting nanocellulose composition.
According to claim 1,
The nanocellulose is a nanocellulose composition for three-dimensional bioprinting, wherein the hydroxyl group on the surface of the cellulose is surface-modified with a carboxyl group.
Low-viscosity nanocellulose manufacturing step; mixing the low-viscosity nanocellulose and an epithelial cell growth factor (EGF) solution; and
Inkjet nozzle ejection step for 3D bioprinting of the mixed solution; A method of manufacturing a nanocellulose composition for three-dimensional bioprinting, comprising a.
7. The method of claim 6,
The low-viscosity nano-cellulose manufacturing step.
preparing a nanocellulose suspension; Nanocrystallization using a grinder or a high-pressure homogenizer; and a step of controlling the viscosity of nanocellulose performed by ultrasonication;
7. The method of claim 6,
The nanocellulose is, the concentration of 1.51% to 1.55% of the total nanocellulose composition, a method for producing a nanocellulose composition for three-dimensional bioprinting.
7. The method of claim 6,
The nanocrystallized nanocellulose has a size of 2.5 nm to 7.0 nm, a method for producing a nanocellulose composition for three-dimensional bioprinting.
7. The method of claim 6,
The low-viscosity nanocellulose is a nanocellulose composition manufacturing method for three-dimensional bioprinting, comprising a carboxyl group of 1.0 mmol to 2.5 mmol per 1 g of nanocellulose.
7. The method of claim 6,
The mixing step of the low-viscosity nanocellulose and the epithelial cell growth factor (EGF) solution comprises a low-viscosity nanocellulose at a concentration of 1.0% to 2.5% and an epithelial cell growth factor solution at a concentration of 0.01% to 0.05% of 2.5 to 1.5: 1. A method for producing a nanocellulose composition for three-dimensional bioprinting, which is mixed at a mixing ratio.
The functional material is supported on the nanocellulose composition for three-dimensional bioprinting prepared according to claim 6, bio-particles.
Filling the 3D bioprinter with the nanocellulose composition for 3D bioprinting prepared according to claim 6; and
3D printing a target tissue-like organ; Containing, organoid (organoid) manufacturing method.
An organoid prepared according to the method of claim 13 .

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