Patent Application
20230266208
The invention relates to tissue homogenizer for processing a sample of biological material, and in particular, a tissue homogenizer which prepares fully disrupted tissue sample first, following allows debris separation from the fully disrupted tissue sample continuously without any disruption.
Molecular diagnostics is dynamic and transformative. It leads to insights in research and treatment in many disease states that are revolutionizing health care. Molecular diagnostics detects and measures the presence of genetic material, including DNA, RNA or proteins that associate with a specific health condition or disease. Tissue sample is a commonly used bio-sample for preparing the genetic material for numerous applications, such as disease diagnostic and therapeutic, cancer research, biomarker research, drug development, etc.
There is a constant need for the development of simplified and high efficiency tissue sample preparation methods which is able to deliver high quality genetic material.
Processing of biological tissue sample is commonly accomplished by mechanical disruption of the tissue to obtain desired small piece of sample or even single cell, following these processed sample or cells are lysed by enzymatic digestion for DNA: RNA or protein extraction and purification. In general, the efficiency and effectiveness of enzymatic digestion are proportional to the surface area of the tissue-enzyme interface. Large surface area can be achieved by mincing the tissue by mechanical means prior to enzymatic exposure.
Some bio-samples, such as bacterial cell, fungi, yeast cell, spore, etc, have strong cell membranes which are very hard to break and are difficult to be processed. These samples have to be dissociated before DNA, RNA or protein extraction and purification.
US Publication No. 20060188892 discloses a kit for preserving DNA, RNA or producing a digested lysate of a tissue sample without homogenizing the sample. The kit requires the use of expensive enzyme chemical and a long incubation time under certain temperature e.g. 40 to 70° C. Suitable container comprising: a buffer; a catabolic enzyme; an ionic detergent and the tissue sample. The container requires complicated one, two or three-dimensional shaking. The maximum amount of tissue sample can be processed by the kit is rather small, only up to 10 mg only. Hence the kit is only suitable for small amount of sample applications, such as diagnosis applications.
Besides use of chemical reagents for tissue dissociation, mechanical force for tissue dissociation is also used. Mechanical force for tissue disassociation is faster than use of chemical reagents hence it is often preferred.
U.S. Pat. No. 7,611,840 discloses a device for sample tissue disruption and/or cell lysis comprising: a piezoelectric material; and at least a second material in contact with the piezoelectric material; the second material has an uneven surface on an opposite side to that in contact with the piezoelectric material, the uneven surface is brought about by a layer of silica beads and contacts a biological sample. The piezoelectric material is actuated by an external voltage source to generate cavitation, which disrupts tissue and/or lyses cells, in particular by a modulated alternative external voltage. The technology is able to process tissue sample in very short time.
Technologies in both US Publication No. 20060188892 and U.S. Pat. No. 7,611,840 have drawbacks, such as the device can only process small amount of sample. However, a large amount of tissue samples, e.g. a few hundred milligrams or even a few grams of sample have to be used for most research application. Although the process time for U.S. Pat. No. 7,611,840 is fast, but the technology cannot separate debris from the disassociated sample.
For using a large amount of tissue samples and reduce time required, U.S. Pat. No. 8,216,528 discloses a sample processing device which crushes a tissue sample to small size by crushing method. In this patent publication, a crushing tool comprises an inner crushing member and an outer side crushing member which is a tubular body capable of housing the inner crushing member therein. The device is configured such that a tissue sample (lymph node) is crushed to a predetermined size, by the inner crushing member of the crushing tool being repeatedly moved upward and downward while being rotated by a motor.
Similar to technology in U.S. Pat. Nos. 8,216,528, 10,173,220 discloses a tissue homogenizer which comprises a housing having a distal end and a proximal end, a cutting blade positioned at the distal end of the housing and a tissue actuator configured to be displaced along a longitudinal axis within the housing. The disadvantage of the homogenizer is that the structure is complicated and the cost is high.
Technologies in both U.S. Pat. Nos. 8,216,528 and 10,173,220 are able to process large amount of sample, but they are unable to separate wanted dissociated solution from unwanted debris.
Filter can be used for separating dissociated solution from debris. U.S. Pat. No. 7,270,284 B2 discloses a tissue homogenizer comprises a first chamber, a pair of blades, a first filter and a second filter. A tissue piece is placed between the first filter and the second filter cut by the blade, and moved by a fluid through the second filter to generate homogenized tissue pieces. The shortcomings of the homogenizer include complicated structure and the chamber of the homogenizer is not easy to be cleaned after use. Thus, cross-contamination of sample may occur. Finally, the tissue homogeniser is not suitable for use in preparing small amount of tissue sample.
Centrifugation also can be used for separating dissociated solution from debris. U.S. Pat. No. 9,962,717 discloses a system, a device and method for combined sample lysis/homogenization and centrifugation for debris separation. The device provides a clear lysate using automated sample grinding, homogenization and lysis of samples of biological and or geological origin, followed by a centrifugation step to clarify the supernatant from solid phase. Both operations (homogenization and centrifugation) are performed within the same sample container and instrument. However, the trajectories of the shaking motions or alternatively of two-dimensional projections of three-dimensional trajectories from using a rotor with swing out sample holders result in a huge amount of heat generated. The higher sample temperature may affect the biological samples for downstream application.
To reduce the temperature of sample during tissue dissociation, U.S. Pat. No. 9,556,410 discloses a homogenization process by employing a blender homogenizing tool with cooling means. The use of blender has many shortcomings, for example, the temperature of the tissue is elevated due to the heat generated by friction between the tissue and the homogenizing tool, or heat generated within the homogenizing tool occurs in the blender operation. There is concern that the proteins contained in the tissue will be thermally denatured when the temperature increases. DNA/RNA will be degraded when the temperature increases also. Therefore, it is suggested that the tissue is cooled within a test tube while being homogenized. There is a constant need for the development of simplified tissue sample preparation methods which deliver high quality and enriched tissue samples. Devices and methods that provide high quality tissue samples with little to no loss (physical or functional) are of interest.
The Inventors have observed a need in the art of sample preparation to have a means for a single step process, with a single piece of equipment and single vial, which may achieve both sample milling/grinding/homogenization/lysis and sample clarification/solid and liquid phase separation, without the process interrupted and without operator influence or intervention. Furthermore, with the increase of scarcity of available lab bench space, it would be beneficial to replace the two instruments commonly employed for lysis/homogenization and centrifugation with one single instrument to perform both functions.
The inventors have observed that the heat generated by the homogenizing devices of the prior art were due to forces generated in 1 Dimension and that a 2 Dimension Impact forces generated by using motion control for generating constant acceleration and deceleration would dissociate tissue sample more effectively.
The inventors have also observed that use of common beads for disassociation of bio-samples could further improve the disassociation process if constant acceleration/deceleration is applied as compared to the variable velocity and acceleration of normal rotary motor generated.
The invention therefore proposes a rotary motor which generates a 2-dimension force for disassociation of bio-sample tissue and then a continuous rotational centrifugation force to efficiently separate the debris and tissue sample. The inventive tissue homogenizer device offers all the benefits not attained by the prior art homogenizer devices.
The use of a rotary motor and a wheel-spoke design housing a plurality of bio-samples offers a low profile homogenizing device which may be readily placed on any table top or flat surface. The tube holders of bio-samples in the wheel-spoke design has a simple loading/unloading mechanism which allows easy fitting of tubes of bio-samples and equally easy removal of the same tubes of bio-samples from these tube holders.
This invention discloses a method and a device for rapid tissue homogenizer, which is capable of processing a large number of samples at one time. The inventive device uses a simple mechanical structure, takes a short processing time, low temperature increase, together with debris separation capability.
A main object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein a combination of dissociation and centrifugation for debris separation is obtained after a tissue sample is fully disrupted or minced.
Still another main object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the homogenizer generates a continuous and high 2 dimension impact force on the tissue sample, resulting a rapid sample disruption process.
Yet a further object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the homogenizer has simple structure and generates low heat and of low temperature, which is good for downstream DNA/RNA, protein process.
Still yet another main object of the present invention is to provide a direct drive tissue homogenizer with debris separation function for preparation of biological material. The homogenizer device comprises: a direct drive motor, a plurality of sample containers, a framework to mount the sample containers. The framework can be a solid bar, a circular solid plate or a circular hollow plate with plurality of arms. The sample container can be sphere, cube, cylinder or any other volumetric shapes that can contain a sample, a plurality of beads and liquid solutions. A plurality of holding slots mounted on the top surface of the framework, wherein the holding slots hold the sample containers; a direct drive motor having a rotating shaft being coupled to a center of a framework and the framework being rotatable about the shaft of the direct drive motor; wherein the direct drive motor generates a motion to the driving arms to produce a horizon motion for dissociation of the tissue in such a way that the beads in the tubular containers cause a blending action on the tissue.
Still another object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the motion generated by the direct drive motor is 20-60 degree reciprocal motion.
Another object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the motion generated has a constant acceleration, and the constant acceleration gives rise to a high impact force on the tissue.
A further yet another object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, further comprises a casing to encompass the drive motor and the framework having mounted with the plurality of container slots thereon.
Another further object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the casing is equipped with a control display for the operation of the homogenizer in preparing a tissue sample.
Still a further object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the tissue sample is being prepared within a time ranging from 10 seconds to 120 seconds, and the requirement of the tissue sample is controlled by the parameter setting on the control display.
Another further object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the beads in the containers are of different sizes together with a specific volume of buffer solution, and the size of the beads used is depending on the kind of tissue sample to be prepared.
Still another main object of the present invention is to provide a method of homogenizing a tissue using a motor driven homogenization device, the method comprises the steps of: supplying to a tubular container a raw material of biological tissue together buffer solution, wherein the container is equipped with a plurality of beads; operating the homogenization device to provide a reciprocating motion to the tubular container; reciprocating of the tubular container at angle of 20-60 degree for 30-120 second; forming a solution in the tubular container; stopping the reciprocating motion of the homogenizer; dividing finely the solution into a dispersion and a plurality of debris to be removed; and collecting the solution as a sample tissue solution.
Another object of the present invention is to provide a method of homogenizing using a direct drive homogenization device, wherein the reciprocating motion of the homogenization device is controlled at the control display on the casing of the homogenization device.
Yet a further object of the present invention is to provide a direct drive tissue homogenizer, wherein a tissue sample includes bacteria cell, fungi, yeast cell, and spore.
A further object of the present invention is to provide a direct drive tissue homogenizer with debris separation capability, wherein the holding slot on the framework holds the sample container by the action of snapping to the container.
It is an object of the present invention to provide a direct drive tissue homogenizer with debris separation capability, wherein the container is equipped with a chip with single or multiple wells.
Other objects and objects and advantages of the present invention will become apparent from the following descriptions, taken in connection with the accompanying drawings, wherein, by way of illustration and example, an embodiment of the present invention is disclosed.
Detailed descriptions of the preferred embodiment are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in virtually any appropriately detailed system, structure or manner.
Each of the containers (20) contain a plurality of beads (22) of varying shapes and sizes. The type of beads (22) and the size of the beads (22) for each disassociation process by the homogenizer (100) depend on the nature of the bio-sample to be disassociated. The container (20) are filled with the beads (22) selected for each disassociation process and a certain volume of water would be then added. Finally a bio-sample of a defined weight is e introduced into the container (20).
Each of the holding slots (14) is spaced apart along the perimeter of the circular framework (12) or the platform, or the like such as a rotary wheel arrangement. Each holding slot (14) holds an individual tubular container (20) in place such that the axis of the containers (20) is in horizontal position, and the circular framework or the platform (12) is rotatable about the shaft (30) of the direct drive motor (10). Each holding slot (14) has a clamp and release mechanism to hold onto the tubular container (20) as the holding of the container (20) is s subjected to a large force as the container spun around at great velocity and at constant acceleration, based on this law of physics:
F (Force)=m (Mass)×a (Acceleration)
wherein F represents Force, m represents Mass and a represents Acceleration.
When the power of the direct drive motor (10) is switched on, the direct drive motor (10) generates a motion to rotate the shaft (30). A horizontal reciprocating motion is produced in the framework/platform (12). The beads (22) inside the tubular container (20) will cause dissociation process to the tissue (not shown) inside the container (20). The purpose of the beads (22) is for the blending action on the tissue. The direct drive motor (10) generates a motion to produce a horizontal reciprocating motion for dissociation of the tissue in such a way that the beads (22) in the tubular containers (20) cause a blending action on the tissue.
A low inertia platform is preferred for the present invention As in
The present invention also relates to a method of preparing homogenized tissue sample using a direct drive motor driven homogenizer (100). The method comprises the steps of: supplying to a container (20) a raw material of biological tissue together with a buffer solution and beads (22); operating the homogenizer to provide a reciprocating motion to the container; reciprocating of the container at angle of ranging from 20-60 degree for a time ranging from 30-120 seconds; forming a solution with biological tissue in the container; stopping the reciprocating motion of the homogenizer; dividing finely the solution into a dispersion and a plurality of debris to be removed; and collecting the solution as a sample tissue solution.
In the preferred embodiment of the present invention, the container (20) is fitted onto the holding slot spaced along the perimeter of the framework. The container filled with beads (22) and buffer solution is then secured onto the circular framework. The direct drive motor of the homogenizer (100) is switched on to provide a reciprocating motion to the tubular container (20). The container is undergone a reciprocating motion at a range of 20-60 degree for 30-120 second. The contents of the bio-sample, and buffer solution form solution in the tubular container (20).
In another preferred embodiment of the present invention, the direct drive homogenizer (100) further comprises a casing (30) to encompass the drive motor (10) and the circular framework (12) having mounted with the plurality of holding slots (14) thereon. The casing (30) is equipped with a control display (34) for the operation of the homogenizer (100) in preparing a tissue sample. The tissue sample is being prepared within a period of time ranging from 30 seconds to 120 seconds of reciprocating motion generated by the direct drive motor (10). There are a plurality of parameters, such as speed, time cycle, etc on the control display (34) to operate the homogenizer (100) and the parameters that are used to prepare a tissue sample are largely based on the requirements for the tissue sample.
To prepare a tissue sample, the container (20) is filled with a plurality of beads (22), which are of different sizes together with a specific volume of buffer solution. The size of the beads (22) used depend greatly on the kind of tissue to be prepared. After a tissue sample in a container (20) is placed into the holding slot (14), the operational parameters, such as number of cycles, speed and time are set. The user would then start the homogeniser which would begin a reciprocating motion of the homogenizer (100). The operations of the homogenizer are controlled at the control display (34) on the casing (30) of the homogenizer (100). The holding slot (14) on the rotary wheel spoke framework (12) holds the container (20) by the action of snapping to the container (20).
The direct drive tissue homogenizer (100) of the present invention can be used to prepare tissue samples, bacteria cell, fungi, yeast cell, and spore. The container (20) can be a standard test tube like structure equipped with an opening and a cap (24) to seal the opening of the tube. The container (20) would be filled with a plurality beads (22) of different sizes (appropriate to the type of tissue to be disassociated) and a certain volume of reagent such as Phosphate Buffered Saline (PBS) or other type of solution.
Again referring to
An user interface in the form of a Display Panel allows an user to set operating parameters such as speed, time of operation and number of cycles. The homogenizer therefore has hardware and software for operational control of the motor including speed, time of operations and number of cycles and so on. The homogeniser may be linked by local networks to computers located outside the laboratory for operational reasons. Operational data such as type of bio-sample tissue to be prepared may be collected and fed back into the homogeniser for more precise operations. The display panel would also have “Start” and “Stop” indicators as well as arrows to move the rotary motor and wheel spoke configuration in increments. Below the rotary motor and wheel spoke configuration is the standalone direct drive Motor. The said framework is mounted on the direct drive motor.
As shown in
Data collected from past operations of the homogenizer would be used to refine the parameters for preparing each type of bio-sample to be disassociated.
The homogenizer of the present invention through a single operation is able to achieve both sample milling/grinding/homogenization/lysis and sample clarification/solid and liquid phase separation, without the process being interrupted and without operator influence or intervention.
The homogenizer of the invention also dissociates large tissue samples more effectively and without the adverse effect of too much heat generated during its operation, which would affect the bio-samples for downstream DNA/RNA and protein process. The outcome is simplified tissue sample preparation process which deliver high quality and enriched tissue samples with little to no loss (physical or functional) to the bio-samples.
The inventive device uses a simple mechanical structure, takes a short processing time, low temperature increase, together with debris separation capability. As the inventive device is equipped with hardware and software for programming and storage of parameters for disassociation and separation of different types of bio-samples, and capability for connection to a network, the homogenizer would be more efficient and superior to the homogenizers currently in use.
The foregoing discussion of the invention and different aspects thereof has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to only the form or forms specifically disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain best modes known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2020/050334 | 6/15/2020 | WO |
