An immunized tumor organoid model and a method for constructing the same

Abstract

The application discloses an immunized tumor organoid and a construction method thereof, and belongs to the technical field of tumor organoids, and the construction method is as follows: under light-proof conditions, a HAMA glue solution containing a photoinitiator is configured, the photoinitiator is added, and mixing is performed to obtain a shell solution; a substrate glue, a culture medium and the HAMA glue are mixed, tumor cells are added thereto, and mixing is performed to obtain a core solution; the shell solution, the core solution and biological oil are respectively injected into a printing chip through a pipeline and discharged through the pipeline, and meanwhile, blue light irradiation is performed to initiate curing, and a double-layer microsphere is obtained; the double-layer microsphere is collected, resuspended and placed in a tumor organoid culture medium for culture, and an immunized tumor organoid is obtained. The method can batch construct tumor organoids under the condition of the same amount of patient-derived tumors, the constructed tumor organoids can simulate a gradual immunization infiltration process of tumor cells and immune cells in the body which is controlled by spatial separation, and the method effectively solves the problem that an immune microenvironment is difficult to reproduce in the construction process of the existing tumor organoids.

Description

Technical Field

[0001] This invention belongs to the field of tumor organoid technology, specifically relating to an immunomodulated tumor organoid model and its construction method. Background Technology

[0002] Malignant tumors pose a serious threat to human health, with their incidence and mortality rates continuing to rise, making them one of the major diseases affecting human health. Although targeted therapy and immunotherapy have enabled precise diagnosis and treatment for most lung cancer patients, the actual number of people who benefit remains very limited. Genetic testing for tumors is the foundation for making precise treatment decisions, but tissue biopsies carry risks and are difficult to repeat multiple times.

[0003] How to effectively establish individualized in vitro drug screening models for patients within a short time window, to screen drugs accurately and in high throughput from existing oncology drugs, to predict drug response in cancer patients, and to provide medication guidance are bottleneck problems that urgently need to be solved in current clinical treatment and research of cancer.

[0004] Three-dimensional structural models derived from patient samples possess genetic lineages and pathological characteristics similar to parental tumors, enabling them to accurately simulate the microscopic morphology and growth of tumors in vivo, making them ideal in vitro tumor models. However, immunogenic in vitro organoid models conventionally rely on vascularization to meet their growth requirements, thus hindering efficient high-throughput in vitro construction. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention provides an immunogenic tumor organoid and its construction method. This method can construct tumor organoids in batches with the same amount of patient-derived tumors, and the constructed tumor organoids can simulate the progressive immune infiltration process of tumor cells and immune cells in vivo, which is controlled by spatial separation, effectively solving the problems existing in the current tumor organoid construction process.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A method for constructing immunomodulated tumor organoids, comprising the following steps:

[0008] (1) Under light-protected conditions, prepare an 8% HAMA gel solution by volume concentration, and then mix it with immune cells by pipetting to obtain the shell solution;

[0009] (2) Take the matrix gel, culture medium and HAMA gel, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare the core solution;

[0010] (3) Using bio-oil as the continuous phase, the outer shell solution, the core solution and the bio-oil are injected into the printed chip through the pipe and discharged through the pipe below the printed chip. At the same time as discharge, blue light is irradiated for 90-120s to obtain bilayer microspheres.

[0011] (4) Collect the bilayer microspheres, resuspend them multiple times in tumor organoid culture medium, and then culture the resuspended bilayer microspheres in tumor organoid culture medium. Change the culture medium every 2 days to obtain tumor organoids.

[0012] Further, in step (1), the volume concentration of HAMA gel in the shell solution is 4%, and the immune cell density is 15,000 cells / μL.

[0013] Furthermore, in step (2), the volume ratio of the matrix gel, culture medium, and HAMA gel is 2:1:1.

[0014] Furthermore, the ratio of immune cells to tumor cells in the outer shell and inner core is 2:1.

[0015] Furthermore, in step (2), the total cell concentration in the kernel solution is 0.75 million cells / μL, and the volume concentration of HAMA gel is 2%.

[0016] Furthermore, the immune cells in step (1) are myeloid immune cells.

[0017] Furthermore, in step (3), the diameter of the outer shell solution channel is 350-400 μm, the diameter of the inner core solution channel is 250-300 μm, and the diameter of the bio-oil channel is 500-600 μm.

[0018] Further, in step (3), the flow rate of the outer shell solution is 11-13 μL / min, the flow rate of the inner core solution is 7-9 μL / min, and the flow rate of the corn oil is 230-270 μL / min.

[0019] Furthermore, in step (4), the bilayer microspheres are suspended in the tumor organoid culture medium and evenly distributed.

[0020] An immunomodulated tumor organoid was prepared using the method described above.

[0021] The beneficial effects of this invention are as follows:

[0022] This method employs a core-shell bilayer heterogeneous HAMA gel structure. The core uses a high-density matrix / HAMA gel to encapsulate tumor cells, mimicking the dense microregions of solid tumors. The shell consists of simple HAMA gel encapsulating immune cells, providing a loose microenvironment conducive to immune cell survival and migration. The two layers form a stable yet permeable barrier through interfacial cross-linking. This structure achieves a mechanical and biochemical gradient structure of "rigid core-flexible shell" through the cross-linking degree and compositional differences between the inner and outer HAMA gel layers. The loose structure of the shell provides immune cells with the low mechanical stress and adhesion signals required for survival, while the core maintains the stable growth of tumor clusters. The bilayer interface in this structure allows small molecules (such as cytokines and metabolites) to diffuse freely, but restricts direct cell incorporation, thus improving culture efficiency.

[0023] In this structure, the outer shell serves as an immune cell chamber, where immune cells can be pre-activated and proliferate. Guided by signals, they gradually migrate inward through the interface, enabling the observation of the regulated immune cell recruitment and killing process, and providing a dynamic model for in vitro immunotherapy research. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the double-layer microsphere structure in this application;

[0025] Figure 2 This is a physical image of the printed chip;

[0026] Figure 3 The image shows a microscopic characterization of the printed microspheres in Example 1.

[0027] Figure 4 The image shows the fluorescence microscopy characterization of the printed microspheres in Example 1.

[0028] Figure 5 This is a bright-field microscopy characterization of the cells encapsulated in Example 1;

[0029] Figure 6 This is a fluorescence microscopy characterization image of the cells in the printed microspheres used in Example 1 for apoptosis detection.

[0030] Figure 7 The image shows a microscopic characterization of the printed microspheres in Comparative Example 1.

[0031] Figure 8 This is a fluorescence microscopy characterization of the cells encapsulated in Comparative Example 2;

[0032] Figure 9 This is a bright-field microscopy characterization of the cells encapsulated in Comparative Example 3. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0034] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0036] The features and performance of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0037] Example 1

[0038] An immunomodulated tumor organoid, the method of which includes the following steps:

[0039] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0040] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0041] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil were printed through the chip channel. The diameter of the outer shell solution channel was 350 μm, the diameter of the core solution channel was 250 μm, the diameter of the bio-oil channel was 500 μm, the flow rate of the outer shell solution was 12 μL / min, the flow rate of the core solution (at 4℃) was 8 μL / min, and the flow rate of the corn oil was 250 μL / min. The microspheres were discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 90 s to obtain a double-layer microsphere.

[0042] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0043] Example 2

[0044] An immunomodulated tumor organoid, the method of which includes the following steps:

[0045] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0046] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0047] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil were printed through the chip channel. The diameter of the outer shell solution channel was 400 μm, the diameter of the core solution channel was 300 μm, the diameter of the bio-oil channel was 600 μm, the flow rate of the outer shell solution was 12 μL / min, the flow rate of the core solution (at 4℃) was 8 μL / min, and the flow rate of the corn oil was 250 μL / min. The microspheres were discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 120 s to obtain a double-layer microsphere.

[0048] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0049] Example 3

[0050] An immunomodulated tumor organoid, the method of which includes the following steps:

[0051] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0052] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0053] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil were printed through the chip channel. The diameter of the outer shell solution channel was 370 μm, the diameter of the core solution channel was 270 μm, the diameter of the bio-oil channel was 550 μm, the flow rate of the outer shell solution was 12 μL / min, the flow rate of the core solution (at 4℃) was 8 μL / min, and the flow rate of the corn oil was 250 μL / min. The microspheres were discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 100 s to obtain a double-layer microsphere.

[0054] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0055] Example 4

[0056] An immunomodulated tumor organoid, the method of which includes the following steps:

[0057] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0058] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0059] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil were printed through the chip channel. The diameter of the outer shell solution channel was 390 μm, the diameter of the core solution channel was 280 μm, and the diameter of the bio-oil channel was 580 μm. The flow rate of the outer shell solution was 12 μL / min, the flow rate of the core solution (at 4℃) was 8 μL / min, and the flow rate of the corn oil was 250 μL / min. The microspheres were discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 110 s to obtain a double-layer microsphere.

[0060] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0061] Comparative Example 1

[0062] An immunomodulated tumor organoid, the method of which includes the following steps:

[0063] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0064] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0065] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil are printed through the chip channel. The diameter of the outer shell solution channel is 350 μm, the diameter of the core solution channel is 250 μm, the diameter of the bio-oil channel is 500 μm, the flow rate of the outer shell solution is 12 μL / min, the flow rate of the core solution (at 4℃) is 4 μL / min, and the flow rate of the corn oil is 250 μL / min. The microspheres are discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 90 s to obtain a double-layer microsphere.

[0066] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0067] Comparative Example 2

[0068] An immunomodulated tumor organoid, the method of which includes the following steps:

[0069] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 15,000 cells / μL immune cell density.

[0070] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 0.75 million cells / μL and a HAMA gel volume concentration of 2%.

[0071] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil are printed through the chip channel. The diameter of the outer shell solution channel is 350 μm, the diameter of the core solution channel is 250 μm, the diameter of the bio-oil channel is 500 μm, the flow rate of the outer shell solution is 12 μL / min, the flow rate of the core solution (at 4℃) is 6 μL / min, and the flow rate of the corn oil is 250 μL / min. The microspheres are discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 90 s to obtain a double-layer microsphere.

[0072] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0073] Comparative Example 3

[0074] An immunomodulated tumor organoid, the method of which includes the following steps:

[0075] (1) Under light-protected conditions, 8% HAMA gel solution was prepared using PBS, and photoinitiator VA-086 was added to it. Then, it was mixed with myeloid immune cells by pipetting to obtain a shell solution with 4% HAMA gel volume concentration and 10,000 cells / μL immune cell density.

[0076] (2) At 4℃, take the matrix gel (Corning commercial Matrigel Matrix), culture medium and HAMA gel in a volume ratio of 2:1:1, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare a core solution with a total cell concentration of 10,000 cells / μL and a HAMA gel volume concentration of 2%.

[0077] (3) Using corn oil as the continuous phase, the outer shell solution, the core solution and the bio-oil were printed through the chip channel. The diameter of the outer shell solution channel was 350 μm, the diameter of the core solution channel was 250 μm, the diameter of the bio-oil channel was 500 μm, the flow rate of the outer shell solution was 12 μL / min, the flow rate of the core solution (at 4℃) was 8 μL / min, and the flow rate of the corn oil was 250 μL / min. The microspheres were discharged through the channel below the printed chip and simultaneously cured by 405 nm blue light for 90 s to obtain a double-layer microsphere.

[0078] (4) Under normal temperature conditions, collect the bilayer microspheres, then centrifuge at 600 rpm / min for 2 min to remove the upper oil phase and PBS solution. The lower bilayer microspheres are resuspended in tumor organoid culture medium multiple times. The resuspended bilayer microspheres are placed in tumor organoid culture medium until the bilayer microspheres are evenly spread in the well plate. The culture medium is changed every 2 days and cultured for 7 days to obtain tumor organoids.

[0079] Test case

[0080] Figure 1 An immunomodulated tumor organoid model; Figure 2 This is a physical image of a printed chip, where Oil is the oil channel, Shell is the shell channel, and Core is the core channel.

[0081] Taking the method in Example 1 as an example, the size of the printed microspheres was measured, as detailed in [link to example]. Figure 3 ; Stain the printed microspheres, see details. Figure 4 The printed microspheres were observed, and the results are shown in the figure. Figure 5 Tumor organoids were cultured, and the survival rate was statistically analyzed. The results are shown in [the table below]. Figure 6 .

[0082] Taking the method in Comparative Example 1 as an example, the size of the printed microspheres was measured, as detailed in [link to example]. Figure 7 Taking the method in Comparative Example 2 as an example, the microspheres were stained and their morphology was observed. See details below. Figure 8 Taking the method in Comparative Example 3 as an example, the printed microspheres were observed. Simultaneously, based on Comparative Example 3, the concentrations of immune cells in step (1) and tumor cells in step (2) were adjusted to 20,000 cells / μL. Specific results are shown in [link to Comparative Example 3]. Figure 9 .

[0083] Figure 3 The results showed that the method in Example 1 could stably generate microspheres of uniform size.

[0084] Figure 4 The results showed that the red part of the microspheres printed by the method in Example 1 was the core part, and the green part was the outer shell part, and the core and outer shell materials were evenly distributed.

[0085] Figure 5 The results showed that the cells inside the microspheres were evenly distributed, maintained a suspended growth state, and were uniform in shape and size.

[0086] Figure 6 The results showed that the microspheres printed by the method in Example 1 maintained high activity after one week of cultivation.

[0087] Figure 7 The results showed that printing using the method in Comparative Example 1 could not stably generate microspheres of uniform size.

[0088] Figure 8 The results showed that when printed using the method in Comparative Example 2, the inner and outer layers of the microspheres were unevenly distributed.

[0089] Figure 9 The results showed that immune cells and tumor cells were printed in a 1:1 ratio, and the uneven distribution and excessive density of cells caused cells to leak out of the microspheres.

Claims

1. A method for constructing immunomodulated tumor organoids, characterized in that, Includes the following steps: (1) Under light-protected conditions, prepare an 8% HAMA gel solution by volume concentration, and then mix it with immune cells by pipetting to obtain the shell solution; (2) Take the matrix gel, culture medium and HAMA gel, mix them to prepare a mixture, add tumor cells to the mixture, mix by pipetting, and prepare the core solution; (3) Using bio-oil as the continuous phase, the outer shell solution, the core solution and the bio-oil are injected into the printed chip through the pipe and discharged through the pipe below the printed chip. At the same time as discharge, blue light is irradiated for 90-120s to obtain bilayer microspheres. (4) Collect the bilayer microspheres, resuspend them multiple times in tumor organoid culture medium, and then culture the resuspended bilayer microspheres in tumor organoid culture medium. Change the culture medium every 2 days to obtain tumor organoids.

2. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, Step (1) The volume concentration of HAMA gel in the shell solution is 4%, and the immune cell density is 15,000 cells / μL.

3. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (2), the volume ratio of matrix gel, culture medium and HAMA gel is 2:1:

1.

4. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, The ratio of immune cells to tumor cells in the outer shell and inner core is 2:

1.

5. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (2), the total cell concentration in the kernel solution was 0.75 million cells / μL, and the volume concentration of HAMA gel was 2%.

6. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (2), the immune cells are myeloid immune cells.

7. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (3), the diameter of the outer shell solution channel is 350-400 μm, the diameter of the inner core solution channel is 250-300 μm, and the diameter of the bio-oil channel is 500-600 μm.

8. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (3), the flow rate of the outer shell solution is 11-13 μL / min, the flow rate of the inner core solution is 7-9 μL / min, and the flow rate of the corn oil is 230-270 μL / min.

9. The method for constructing immunomodulated tumor organoids as described in claim 1, characterized in that, In step (4), the bilayer microspheres are suspended in the tumor organoid culture medium and evenly distributed.

10. An immunomodulated tumor organoid, characterized in that, It is prepared by any one of claims 1-9.

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