TW550066B - Micro bio-cultivation device - Google Patents

Abstract

A micro bio-cultivation device provides an implantable bio-artificial micro device installed with ""cell apartments"" for grazing (planting, cultivating, releasing hormone) insulin-releasing Islets of Langerhans or other similar hormone secreting cells or tissue. Two sides of the cell apartments are installed with a dynamic micro-electric field array filter which includes a dense electrode array, and a micro fluid driving device which periodically alternates its driving direction to form a reciprocating body fluid flow in the micro channel so that all fluid flow entering the device after going through an immune isolation of the micro-electric field array; all fluid flow that discharges from the device accelerates the release of the cell secretion under the driving of the micro fluid. The invented dyamic micro-electric field array filter system provides a physical immune protection to protect the planted cells from the attack of the host immune system, thereby increasing the survival rate of the cells and increasing the physiological functions thereof.

Description

550066 A7

V. Description of the invention (/) [Application scope of the invention] The present invention relates to a miniature biological cultivation device, in particular to a miniature biological cultivation device with a function of filtering small and harmful substances. The invention also discloses the structure of the miniature biological cultivation device. And manufacturing methods. [Background of Invention] Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs According to forecasts, the world ’s diabetes population will increase from 125 million to 300 million from 1995 to 2025, and most patients will reach the age of 60 Above the age. The IPTR (International Pancreas Transplant Registry) reports: As of December 1997 ’there were approximately 11,000 SPK (simultaneous pancreas plus kidney transplants) cases worldwide. Among them, the first year survival rate was 94%, and the fifth year survival rate was still as high as 80%. Therefore, as long as supplemental healthy islet cells enter the patient's body, diabetes may be effectively controlled. Although the most effective method for replenishing islet cells in patients is pancreas transplantation, pancreas transplantation involves large-scale and high-risk surgical surgery, and it is very easy to have acute immune response (HAR, Hyperacute rejection), and it is healthy at the same time. The amount of pancreatic donation cannot meet the needs of the majority of diabetic patients. Therefore, Xenograft islet transplantation, such as transplantation of healthy porcine pancreatic islets into the human body, has become an effort of the global medical community. direction. This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm). Please read the back of the © © before filling in the line 550066 A7

V. Description of the invention (Legal) Only through close contact with the Tengdao cells must the acid solution be verified by animal experiments, can the blood glucose concentration change be accurately monitored and insulin secreted and transported throughout the body cells in time. However, if the characters in the different view are in close contact with the host's blood, they will face the most severe immune response attack (the immune response dominated by CD4helperTCell). Therefore, how to provide perfect immune protection for the implanted cells is the most difficult technical bottleneck for islet cell transplantation in xenogeneic animals, and it is also the reason why there are no successful examples in the world. In the conventional technology, the application of "APA encapsulation by alginate / poly-L-lysine / alginate" to islet cells is the most mature means of immune protection development.

Studies have shown that the isolated islet population after APA encapsulation is still alive in the host (Ratislets to NOD mouse) on day 80, but has gradually collapsed and broken down since then. The University of California, Berkeley, Ferrari and others first published the technology of microfabricated Immunoisolation membranes in 1998, and actually used in vitro cell culture to isolate the chip Membrane does resist the invasion of immunoglobulins (IgG) (see ΤΑ Desai, DJ Hansford, WH Chu, T Huen, M Ferrari: "Investigation islet immunoisolation parameters using microfabricated membranes", Mat · Res · Soc · Sym · Proc · Vol · 530, 1998) 〇4 This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)

Order --------- Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 550066 A7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs ______ Β7 ___ 5. Description of Invention ($) Leap Year (1999) The Research Group Further published technical details: The Sandwiched Polysilicon / oxide / P · polysilicon layer was used to prepare a "micro-machined nanofiltration layer". This technology can accurately construct pores with high mechanical strength and consistent size. To 10 nm (see: WH 〇1 Tun R Chin, T Huen, M Ferrari: ^ Silicon membrane nano filters from sacrificial oxide remoral, "J. Microelectromechanical Systems, Vol. 8, No. 1, 1999) An organ (Vascular bioartificial organ, such as U.S. Patent No. 5,534,025, 1996) is an improved structure of an islet implant. The focus of this patent is to place islet cells in the arteriovenous space to closely contact the host's blood and perform its physiological functions. However, this patent does not reveal the technical details of the immune isolation membrane between Tengdao and Su and the blood. Bioartificial pancreas published by Nier et al. (Bioartificial Pancreas US Patent No. 5,855,616, 1999) is another novel design, focusing on the tubular structure that houses the islets, and surrounding a layer of fibers that infiltrate the cell growth factors in advance. Hope After implantation, the device can stimulate the growth of host cells and integrate them, thereby providing immune isolation and avoiding blood clotting. However, whether the rate of host cell growth is too late to prevent the immune system from attacking internal islet cells Doubt. In the conventional technology, the research and development direction of immune isolation is to narrow the gap 5 3 | Μ 1 Read the back of I * Note I mean I matter I item I then I fill in m pages II% order

550066 Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of invention (per) The cut-off pore size of the membrane. Generally, the cutoff pore size of Microcapsule is 10 ~ 30nm. Micro-machined nano-filter layers can be as small as 10 nm. Such pores can roughly block molecules with a molecular weight of 100 to 200 kDa. Using a denser high-molecular membrane Poly-L-Lysine (PLL) Layer can block molecules above 60kDa. For the representative role of the immune system: Lymphocyte (lymphocytes) or immunoglobulin IgG (50kDa), already has a good immune isolation effect. Unfortunately, recent studies have shown that those who do great harm to implanted cells are smaller substances, such as cytokines (Cytokine, 15-25kDa) and chemokines (Chemokine, 8kDa). Among them, the interleukin-1β (Interleukin-1β) involved in the immune mechanism dominated by CD4 helper T cell will not only promote the implanted cells to produce Cytotoxic aldehydes (Cytotoxic aldehydes), but also will trigger the programmed cell death (Apotosis) . On the other hand, although the important hormone prepared by the implantation of Tengdao cells: insulin (Insulin), although the molecular weight is 6kDa, it is likely to still combine with C-peptide of similar volume and maintain the Proinsulin state when the cell releases it. . Its volume is very close, even larger than cytokines or chemokines. The result is a serious logical dilemma: it is likely that what is expected to be blocked is smaller than what needs to be released. Therefore, if only the traditional concept of the filter layer is used, even the ability to repeatedly reduce the pore size of the barrier will not help. ------------ (Please read the notes on the back before filling this page)

Order --------- Line I Printed by Consumer Cooperative 550066 5. Description of the Invention (5) Therefore, it is necessary to provide a new immune protection device that can separate the blood flow (jnlet) from the graft and the outflow. Graft blood flow (〇utlet), so that each has different release standards:

Inlet: WBC, Antibody, Cytokine, Chemokine cutoff

Glucose, Oxygen, Nutrient pass

Outlet: Retrovirus, Antigen cutoff

Insulin, Glucagon pass Note: Retrovirus is a rnA virus that is harmless to the host and harmful to the host. It must be prevented from infecting the host with implanted cells. [Objective of the Invention] The object of the present invention is to provide a novel micro-biological culture device, which can give different release standards to the microfluids flowing into and out of the micro-organisms. Thread The object of the present invention is also to provide a micro-organism culture device which can filter harmful substances but can release harmless substances or beneficial substances. The object of the present invention is also to provide a miniature biological culture device having an electrode array immune blocking function. The object of the present invention is also to provide a method for manufacturing the above-mentioned micro-biological culture device. [Brief description of the invention] This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 public love) 550066 A7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (^) The microbiological culture of the invention The device provides an implantable bio-artificial micro device that can be implanted in animals. A "cell apartment" is built in the device, which can be used for grazing (implanting, cultivating, and hormone releasing). Islets of Langerhans) or other similar endocrine organism cells or tissues. "Dynamic micro-electric fidd array filter" is set on both sides of the cell apartment, which includes a densely arranged micro-pipe array; micro-electrodes are arranged across each pipe, and the electrode polarity is switched alternately periodically. A reciprocating body fluid flow is generated inside the microchannel, and all the body fluid flows entering the device first pass through the immune block of the micro electric field array; and all) body fluid flows out of the device are accelerated by the release of cell secretions under the drive of microfluidics . The "Dynamic Micro Electric Field Array Filtering System" of the present invention provides a physical immune protection barrier to resist the host immune system's attack on the implanted cells, improve the cell survival rate and exert its physiological functions. The above and other objects and advantages of the present invention will be made clearer by the following detailed description and reference to the following drawings. [Explanation of the drawings] Fig. 1 shows a schematic plan view of the miniature biological culture device of the present invention. Figures 2 (a)-(f) show the production flow of the micro-biological culture device of the present invention 8 size coffee x 297 public love) equipment -------- order ----- (Please read the note on the back first Please fill in this page for matters) # 1 550066 Printed A7 by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs -----------------__ 5. Description of the invention (7) Process chart. [Detailed description of the invention] FIG. 1 shows a schematic plan view of the miniature biological culture device of the present invention. As shown in the figure, the micro-biological culture device of the present invention has a micro-tube (丨), which is fabricated on a substrate (not shown) for fluid (such as blood) to pass through. A culture module structure (6) is provided in the pipe d) to accommodate the cultured biological cells (7). In the present invention, two types of electrode power pumping principles are used to separately process the blood flow in and out of the device. The blood flow can be electro-hydrodynamic, and the blood flow can be electro-osmosis. The electro-hydraulic power pump, which is the fluid power source entering the cultivation module structure (6), is a first group of electrode arrays (2) (3) arranged very close to each other in a small pipe (1). The positive electrode (3) is dispersed into a columnar array and penetrates into the pipeline, and the negative electrode (2) is also a columnar array into the pipeline, but is staggered with the positive electrode array (3). In other examples, electrode group, electrode plate group or fork electrode can be used. After the voltage is applied, the positive and negative electrodes (2) and (3) can form an electron flow circuit through the intervening fluid, and at the same time, drive the surrounding fluid to flow in the direction of the electron flow, forming an electro-hydraulic effect (EΗ〇 pumping). At the same time, a micro-electric field formed between the positive and negative electrode arrays is used at the same time to form a capture network 'to intercept negatively charged molecules such as IgG.Cytokine, Chemokine. The size of this electro-hydraulic paper is applicable to China National Standard (CNS) A4 (210 X 297 public love) _ · clothing -------- order --------- line one (please read first Note on the back, please fill out this page again) 550066 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 — · — B7_ V. Description of the invention (and) Power pump provides a micro electric field that can block tiny particles, and there is a micro electric field Generate the utility of the device. In terms of the liquid power source flowing out of the cultivation module structure (6), the present invention arranges a second group of electrodes (4) (5) on the wall of the micro-pipe (1), and is flatly attached to the pipe (1) in the form of a sheet electrode On the wall. After the electrode (4) is turned on for the negative voltage and the electrode (5) is turned on for the positive voltage, the electrical polarization of the pipe wall is first caused, and a local potential gradient is formed. At this time, the charged particles in the fluid are affected by this potential gradient, which can drive the blood flow from the electrode (4) to the electrode (5), forming the electroosmosis effect (E0 pumping). This effect is used to provide a driving force to quickly release the insulin released by the intervening Tendo cells (7). This electroosmotic driving force can be used as the main driving force of blood flow and become the driving device of microfluidics. The culture module structure (6) of the present invention is to precisely define a rigid geometric structure in the middle of the micro pipeline (1) by using a micro-machining process to form a so-called cell apartment (6), so that the cultured organism (7), such as Islet cells are placed in the apartment (6). In application, the surface of the cell apartment (6) can be coated with biocompatible materials, such as paryleneC and the like. Two sets of electro-hydraulic power electrodes (2) (3), (8) (9) and two sets of electro-osmotic electrodes (poXioMu) are arranged across the cultivation module structure (6) on both sides. A controller (not shown) can be used to switch the four groups of electrodes on and off to generate different fluid flows. 10 Paper sizes are applicable to China National Standard (CNS) A4 ^ 72_10 X 297 mm1 "

-------- Order ------- (Please read the phonetic on the back? Matters before filling out this page) 550066 A7

550066 A7 B7 Employees of the Intellectual Property Bureau of the Ministry of Economic Affairs. Co-printed by the cooperative. V. Invention Description (丨 0 direction. All blood flows into the device, no matter which direction they enter, are electro-hydraulic before they come into contact with islet cells. The micro-field of the electrode array is immune-blocked; and all the blood flowing out of the device, regardless of the direction from which it flows, can accelerate the smooth release of membrane islands. Figures 2 (a)-(f) show the miniature biological culture device of the present invention As shown in the figure, the micro-biological culture device of the present invention can be prepared according to the following steps: (a) Wafer fabrication: silicon substrate is used for deep micro-channel etching. Silicon nitride (SiN4) is used as a cover The mask layer is etched with KOH, THAM (tetramethyl ammonium hydroxide) or EDP (ethylene diamine pyrozine) solution. Etching to the required depth (200-300um). (B) Fabrication of electrode and culture module structure: The silicon nitride masking layer is removed with hot phosphoric acid, and chromium (Cr) and gold (Au) are sputtered as a seed layer of the electrode array electron microscope, and then rotated Spinningcoating — a thick photoresist layer. This layer must completely cover the seed layer. After the lithography process is used to make the array module structure array pattern, gold plating is performed. The plating height is about one-half to one-third of the micropipe. I. (c) Electro-hydraulic power electrode formation ... Remove the thick photoresist layer with hot sulfuric acid, and then use 12 paper sizes to apply Chinese National Standard (CNS) A4 specifications (210 X 297 mm) ------- -^ ---------. (Please read the precautions on the back before filling this page) 550066 A7

V. Description of the invention (丨 丨) Printed and coated with a thick photoresist layer by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, this layer also needs to completely cover the seed layer; For gold plating. (d) Formation of electroosmotic electrode ... Remove the thick photoresist layer with hot sulfuric acid, and then spin-coat a thick photoresist layer again. This layer needs to completely cover the electrode array; the electroosmosis (EO) electrode pattern is made by the lithography process After the 'Luo (Cr) and gold (Αυ) the rest. (e) Insulating layer formation: After removing the thick photoresist layer with hot sulfuric acid, cover the insulating layer and deposit the Paiylene polymer insulating layer (π) by the vapor deposition method. This method can conformally deposit high-depth patterns. )effect. (f) Covering: Cover the drilled glass (18) (leave it on the wire pad or insert the cell), and perform anodic bonding with this wafer. [Effects of the invention] The rigid microstructure completed by the semiconductor process has excellent consistency and precisely defined geometric dimensions. It can build the optimal culture module structure (cell apartment) configuration according to the biological characteristics of the bearing cells and enhance the cells. Stability and stability of physiological functions. The invention separates the inflow and the bleeding flow separately, and any blood flow passes through the micro-electric field of the electro-hydraulic power electrode array before being contacted with the cells, and all the blood flows out of the device are driven by the effect of electroosmosis. Irrelevant, it can form no 13 ---------------------- Order ----- (Please read the precautions on the back before this page) Line ' Standard of this paper standard (CNS) A4 (210 297 mm) 550066

The same small molecule release standard printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs broke through the logical dilemma of the aforementioned cytokine (which needs to be blocked outside the device) being smaller than insulin (which needs to be released from the device). The invention can periodically and alternately switch the flow direction during operation, which can prevent any blood cells, proteins, anti-thorium and cytokine molecules from accumulating at the entrance due to being blocked by the micro-field immune blockage; it can also accelerate the supply of cell nutrients, waste removal and evacuation Fast release of island hormones. In the example of the present invention, a unidirectional microfluidic driving device is used, but if a microfluidic driving device is used, it is not necessary to provide two sets of driving devices. In addition, even if it is a unidirectional driving device, it is not limited to driving by electroosmosis. The invention can also produce the special effect of preventing blood cells (or fibrin) from sticking to the device: the blood flow side must be the starting edge of the EHD negative electrode at any time, generating a Coulomb repulsion (Electrical rejection) to prevent blood cells from approaching; and bleeding Although the E0 positive electrode is activated on the flow side, the substantial blood ejection (current rejection) can also push away the blood cells. In the above description, the culture of cells in blood is taken as an example. However, the present invention can be applied to various fluids, and is not limited to blood or human blood; the cultured cells are not limited to animal cells, and other cells such as plant cells, strains, viruses, and even chromosomes and DNA fragments can be applied. 14 This paper size applies to China National Standard (CNS) A4 specification (210 X 297 public love) ▼ Shang ------ (Please read the precautions on the back before filling this page) Order ----- Thread 550 066 A7

Claims (1)

550066 A8 B8 C8 D8 Printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Patent application scope 1. A miniature biological culture device, including: a microfluidic channel for a microfluid to pass through; a biological culture module structure located in the Inside the microchannel, it is used to contain the biological cell or part of the cell to be cultured, and can allow the microfluid to flow through the biological cell or part of the cell; a set of micro-electric field generating device is located in the microchannel, including most The positive electrode and most of the negative electrodes, when voltage is applied at the same time, can generate an electronic flow circuit between the microfluids and form a micro electric field; and a set of microfluidic driving devices located in the microchannel to generate a driving force To drive the microfluid to move within the microchannel; characterized in that the micro-electric field generating device is located on the upstream side of the microfluid under the microfluid driving device. 2. The device according to item 1 of the scope of patent application, which includes two sets of micro-electric field generating devices, which are respectively located in the microfluidic channel, opposite to the two sides of the biological culture module structure. 3. If the device in the scope of patent application is No. 1, the microfluidic moving device is a unidirectional driving device, and includes two sets of driving devices, which are respectively located in the microfluidic channel, opposite to the two sides of the biological culture module structure. 16 This paper size applies to China National Standards (CNS) A4 ¥ ⑵G x 297 public hair 5 -----* * --------------------- Order --------- line (please read the note on the back? Matters before filling out this page) 550066 Employees of Intellectual Property Bureau of the Ministry of Economic Affairs printed a patent application patent park 4. If the scope of patent application is the second The device of item, wherein the microfluidic driving device is a unidirectional driving device, and includes two sets of driving devices, which are respectively located in the microfluidic channel, opposite to two sides of the biological culture module structure. 5. The device according to item 2 or 4 of the scope of patent application, in which any one of the two sets of micro-electric field generating devices is on the upstream side of the microfluid, and the on-voltage is initiated. 6. The device according to item 4 of the scope of patent application, further comprising a control device for periodically driving a set of one-way microfluidic moving devices and a set of micro-electric field generation on the upstream side of the driving direction of the microfluidic driving device Device, and another set of unidirectional microfluidic driving devices and another set of micro-electric field generating devices. 7. The device of claim 2, 2, 3 or 4, wherein the structure of the biological cultivation module has a rigid geometric structure. 8. The device according to item 7 of the patent application scope, wherein the surface of the biological culture module is coated with a biocompatible material. 9. The device according to the scope of the application for patent item ^^ or 彳, wherein the micro-field generating device comprises at least one column of positive electrode column groups and at least one column of negative electrode column groups. 10. If the device in the scope of patent application No. 2, 3 or 4 is applied, wherein the microelectronics 17 (CNS) Α4 specifications (21〇ό (factory, f please K. ^ Α read it back; ^ 意 _ ^ 、 ^ Fill in 5 ”'^. Stomach) 550066 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Βχ v 's 18.6. Patent Application Park — ^ 一' --- The field generating device includes at least one column of positive electrode plate groups and at least one column of negative electrode plate groups. 11. · A method for manufacturing a micro-biological culture device, comprising: preparing a substrate; forming a microfluidic channel in the substrate; forming a micro-biological culture module structure in the microfluidic channel and at least one micro-electric field generating electrode group A seed layer; forming at least one set of micro-electric field generating electrode groups in the microfluidic channel; each including at least one set of positive electrodes and at least one negative electrode; and forming at least one set of microfluidic driving electrodes in the microfluidic channel, each It includes at least one positive electrode and one negative electrode. 12. The method according to item 11 of the scope of patent application, wherein the micro-electric field generating electrode group forms two groups, which are respectively located on two sides of the biological culture module structure. 13. The method according to item 11 of the scope of patent application, wherein the microfluidic body electrode group forms two groups, which are respectively located on two sides of the biological culture module structure. 14. · The method described in the scope of patent application No. 11 顼, including the application of the Chinese National Standard (CNS) A4 specification (/ 2 (factory spring (please ^ wen feed back the shell of the · .t 荠 fill pain ts) 550066 Bs (-S ih VI. The step of applying a biocompatible material to the surface of the structure of the cultivation module structure for a patent application. 15. The method described in item 14 of the patent application scope, wherein the biocompatible material is parylene C. 16. The method according to item 12 of the patent application, wherein the electrode system of the micro electric field generating electrode group is formed in a columnar shape. 17. The method according to item 12 of the patent application, wherein the micro electric field generating electrode group is The electrode system is in the form of a sheet. Sni c: \ my ckxmments \ tracy \ itri03sp-OB09 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 9 11 This paper size applies the Chinese National Standard (CNS) Λ · 4 Specification (2 丨 0 > 297 Mm)