TW201329231A - Method and device for isolation of non-fat cells from an adipose tissue - Google Patents

Abstract

The present disclosure is related to a method and a device for isolating non-fat cells from adipose tissue samples collected from animal or human sources.

Description

Method and device for separating non-fat cells from adipose tissue

The present invention relates to a method and apparatus for isolating non-fat cells from an adipose tissue sample collected from an animal or human source.

Many techniques in biology and medicine, such as cell separation, flow cytometry, cell analysis, and cell therapy rely on dissociating tissue to separate individual cells. Processing tissue samples often involves a variety of actions such as shredding, washing, enzymatic digestion, dissociation, incubation, mixing, block and debris removal, and concentration. In research laboratories, these actions are often performed manually, allowing the sample to be transferred from the test tube to the test tube in an open environment. Such manual handling requires highly trained personnel and the manipulation of biological samples poses a potential contamination and infection risk.

Recently, there has been interest in separating cells from adipose tissue in research and medical communities. Several techniques have been developed to safely remove portions of adipose tissue from a patient or animal. For example, tumescent liposuction and water jet liposuction techniques have been widely used to remove adipose tissue from a patient. Adipose tissue contains fat cells that store fat and other non-fat cells that maintain tissue. The constituent cells of adipose tissue, their effects and their interactions have not been completed Fully understood and the subject of active academic and clinical research.

To study adipose tissue, it may be necessary to dissociate the tissue and isolate the constituent cells. Treatment can include releasing constituent cells, removing debris and unwanted cells, concentrating and enriching related cells, and washing cells. This process can be laborious and may require highly trained operators, expensive instrument settings, and laboratories with appropriate biosafety measures. A variety of manipulations can also result in significant loss of associated cells, making separation of rare materials and low-popular cells difficult and unreliable. In addition, the risk of cross-contamination and infection can be substantial when using human samples.

There is therefore a need to obtain a method for efficiently isolating related cells (especially cells which are not fat cells) from adipose tissue, and to obtain a device which makes it easy and safe to separate non-adipocytes from adipose tissue.

Bags comprising flexible plastic sheets are widely used to collect, process and store biological tissue samples such as peripheral blood, cord blood, blood components, plasma, bone marrow, lipoaspirate, and the like. The bag has the advantage of being flexible and expandable and is capable of changing its internal volume to accommodate different volumes of sample. To aid in sample handling, many individual bags are often fluidly connected using an outer tube to form a system. This bag and tube system has been widely used in sample processing (eg, blood fractionation, cell separation, etc.). However, when more processing actions are integrated, the bag and tube system quickly becomes complex, difficult to use, and difficult to manufacture. These systems become insulating sleeve-like and become prone to kinking, with many components shaking from each other. In order to use such devices, the operator needs a higher level of training and long-term hands-on time to set up complex devices. The operator also needs to be in the correct order Install additional parts in the correct position for extra attention. In addition, because multiple bags, components, and pipe sections often have to be separately fabricated and then assembled, such devices and systems can be difficult to manufacture and expensive to manufacture, and such devices can be labor intensive and can exhibit leakage and contamination ( The risk of a system failure. For clinical applications, these devices are often used in a single use and their reliability is important. The risk of intensive labor and equipment failure is a major obstacle to conventional insulated casing-like systems.

According to one aspect of the present disclosure, an apparatus for separating non-fat cells from adipose tissue samples is provided. The apparatus includes a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers including a sample dissociation chamber, the sample The dissociation chamber includes an inlet and an outlet; a waste collection chamber including an inlet in fluid communication with the outlet of the sample dissociation chamber; and a cell refining chamber including an inlet in fluid communication with the sample dissociation chamber, And an exit.

According to some embodiments, the sample dissociation chamber further includes a mesh filter comprising pores having a pore size between 70 μm and 300 μm .

According to some embodiments, the apparatus further comprises a mesh filter included in the cell refining chamber, the mesh filter comprising pores having a pore size between 20 μm and 50 μm .

According to some embodiments, the sample dissociation chamber further comprises a first screen a filter, the first mesh filter comprising a hole having a first aperture, and wherein the cell refining chamber further comprises a second mesh filter, the second mesh filter comprising a hole having a second aperture, wherein the second The aperture is smaller than the first aperture.

According to some embodiments, the apparatus further includes a member that controls the fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell refining chamber.

According to some embodiments, the member that controls the fluid connection includes a piston.

According to some embodiments, the apparatus further includes a flow control device configured to introduce at least one of the irrigation fluid and the dissociation fluid into the sample dissociation chamber and having an outlet in fluid communication with the sample dissociation chamber.

According to some embodiments, the apparatus further includes a member that applies pressure to one of the sample dissociation chamber and the cell refining chamber.

According to some embodiments, the apparatus further comprises a downstream processing device in fluid communication with the outlet of the cell refining chamber and comprising at least one microfluidic device configured to direct the fluid output from the cell refining chamber Separating into a first solution having a first concentration or a plurality of related cells and the second solution having the concentration of the one or more related cells less than the concentration of the first solution, wherein the relevant cells comprise Non-fat cells isolated from adipose tissue samples.

According to one aspect of the present disclosure, a sterile and substantially separate adipose tissue treatment system is provided. The system includes an organization processing room, the group The woven processing chamber includes an inlet, an outlet, and at least one screen filter disposed between the inlet of the tissue processing chamber and the outlet of the tissue processing chamber; the waste collection chamber, the waste collection chamber and the tissue The processing chamber is included in the same housing, the waste collection chamber including an inlet in fluid communication with the outlet of the tissue processing chamber; and one of: a debris removal chamber including a debris removal mechanism; and a sample collection chamber The sample collection chamber and the tissue processing chamber are included in the same housing and are in fluid communication with the tissue processing chamber.

In accordance with one aspect of the present disclosure, a method of processing a fatty tissue sample in a tissue processing system is provided. The method includes introducing a sample of the adipose tissue to be treated into the first chamber via the inlet of the first chamber, processing the adipose tissue sample in the first chamber, and passing the cells from the first chamber through the outlet of the first chamber The inlet of the two chambers is transferred to a second chamber that is included in the same housing as the first chamber.

According to some embodiments, processing the adipose tissue sample comprises dissociating the adipose tissue sample.

According to some embodiments, processing the adipose tissue sample comprises removing excess fluid from the adipose tissue sample in the first chamber.

According to some embodiments, processing the adipose tissue sample comprises washing the adipose tissue sample with a rinse solution in the first chamber.

According to some embodiments, processing the adipose tissue sample comprises washing the adipose tissue sample with a rinse solution in the first chamber, and dissociating the adipose tissue sample using a dissociation solution comprising at least one enzyme in the first chamber.

According to some embodiments, the dissociation fluid comprises collagenase.

According to some embodiments, the dissociation fluid comprises collagenase, deoxyribonuclease, and hyaluronidase.

According to some embodiments, dissociating the adipose tissue sample using the dissociation fluid occurs at about 37 degrees Celsius.

According to some embodiments, the method further includes removing debris using a mesh filter included in the second chamber.

According to some embodiments, the mesh filter has a pore size between 15 microns and 100 microns.

According to some embodiments, the method further comprises trapping the sample in the first chamber and transferring the waste liquid through the screen filter included in the first chamber and the first outlet of the first chamber via the inlet of the third chamber To the third chamber in the same housing as the first chamber.

According to some embodiments, the method further comprises using a microfluidic device to augment the non-fat cell population.

According to some embodiments, the non-fat cells comprise stem cells.

According to some embodiments, the method further comprises collecting cells from the tissue processing system.

According to some embodiments, the method further comprises processing the cells in a downstream processing device in fluid communication with the outlet of the second chamber and including at least one microfluidic device configured to separate the cells into the first solution and the second solution, The first solution has a first concentration of non-fat cells and the second solution has a concentration of non-fat cells that is less than the concentration of the first solution.

According to some embodiments, the collected cells are stromal vascular fraction cells.

According to one aspect of the present disclosure, an apparatus for separating cells from adipose tissue is provided. The apparatus includes a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material, the plurality of chambers The sample dissociation chamber includes an inlet and an outlet; a waste collection chamber including an inlet in fluid communication with an outlet of the tissue dissociation chamber; and a cell refining chamber including a sample and a sample The outlet of the dissociation chamber fluid communication, and an outlet.

According to some embodiments, the sample dissociation chamber further comprises a filter screen.

According to some embodiments, the apparatus further comprises a filter screen, the filter screen being included in the cell refining chamber.

According to some embodiments, the sample dissociation chamber further includes a first filter screen, the first filter screen including a hole having a first aperture, and wherein the cell refining chamber further comprises a second filter screen, the second filter screen comprising A hole having a second aperture.

According to some embodiments, the second aperture is smaller than the first aperture.

According to some embodiments, the apparatus further includes a member that controls the fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell refining chamber.

According to some embodiments, the member that controls the fluid connection includes a piston.

According to some embodiments, the apparatus further includes a flow control device configured to introduce at least one of the irrigation fluid and the dissociation fluid into the sample dissociation chamber and having an outlet in fluid communication with the tissue dissociation chamber.

According to some embodiments, the apparatus further includes a member that is directed to the sample

According to some embodiments, the apparatus further comprises a downstream processing device in fluid communication with the outlet of the cell refining chamber and comprising at least one microfluidic device configured to direct the fluid output from the cell refining chamber Separating into a first solution having a first concentration or a plurality of related cells and a second solution having the concentration of the one or more related cells that is less than the concentration of the first solution.

In accordance with one aspect of the present disclosure, a substantially isolated adipose tissue processing system is provided. The system includes an adipose tissue processing chamber including an inlet, an outlet, and at least one filter screen disposed between the inlet of the tissue processing chamber and the first outlet of the tissue processing chamber; a collection chamber, the waste collection chamber and the tissue processing chamber being included in a same housing, the waste collection chamber including an inlet in fluid communication with the first outlet of the tissue processing chamber; and one of: a debris removal chamber, the debris removal The chamber includes a debris removal mechanism, and a sample collection chamber, the sample collection chamber and the tissue processing chamber being included in the same housing and in fluid communication with the second outlet of the tissue processing chamber.

According to some embodiments, the system further includes a fluid volume measurement chamber in the same housing as the adipose tissue processing chamber and including an inlet and an outlet in fluid communication with the inlet of the adipose tissue processing chamber.

According to some embodiments, the inlet of the fluid volume measuring chamber and the volume of the fluid The outlets of the chambers each include a check valve.

According to some embodiments, the first outlet and the second outlet comprise an outlet of a piston in fluid communication with the adipose tissue processing chamber.

According to one aspect of the present disclosure, a method of isolating cells from adipose tissue is provided. The method includes introducing a sample to be treated into the tissue processing chamber via an inlet of the tissue processing chamber, processing the sample in the tissue processing chamber, and releasing the cell from the tissue processing chamber through the outlet of the tissue processing chamber to the tissue processing chamber via the inlet of the sample storage chamber Included in the sample storage compartment in the same housing.

According to some embodiments, the method further comprises trapping the cell sample in the tissue processing chamber and transferring the waste liquid to the tissue via the screen outlet of the screen filter and the tissue processing chamber included in the tissue processing chamber via the inlet of the waste collection chamber The processing chamber is included in a waste collection chamber in the same housing.

According to some embodiments, the method further comprises extracting a cell sample from the tissue processing system.

According to some embodiments, the method further comprises processing in a downstream processing device in fluid communication with the outlet of the sample storage chamber and including at least one microfluidic device configured to separate the extracted cell sample into a first solution and a second solution The extracted cell sample, the first solution having one or more associated cells at a first concentration and the second solution having the one or more associated cells at a concentration less than the concentration of the first solution.

According to some embodiments, processing the tissue sample in the tissue processing chamber comprises introducing the tissue cleaning fluid into the tissue processing chamber.

According to some embodiments, processing tissue samples in a tissue processing chamber further This includes introducing tissue dissociation fluid into the tissue processing chamber.

According to one aspect of the present disclosure, a method of isolating cells from adipose tissue is provided. The method includes introducing a sample of lipoaspirate to be treated into a chamber comprising a screen, removing excess fluid from the sample, passing the dissociated sample through at least one screen to remove fat cells and debris, and substantially shifting from the sample The samples were concentrated at least three times in addition to red blood cells and using a microfluidic device.

According to some embodiments, the method further comprises washing the sample with a washing liquid.

According to some embodiments, the method further comprises mixing and cultivating the sample with a dissociating solution comprising at least one enzyme.

According to some embodiments, the microfluidic device comprises a plurality of microfluidic channels having a substantially constant depth and at least one dimension less than 750 microns, wherein at least two microfluidic channels are disposed on one surface of the substrate .

According to some embodiments, the dissociation fluid comprises collagenase, and wherein the sample and the dissociation fluid are incubated between about 20 degrees Celsius and about 40 degrees Celsius.

According to some embodiments, the dissociation fluid comprises collagenase and deoxyribonuclease, and wherein the sample and the dissociation solution are incubated at about 37 degrees Celsius.

According to some embodiments, the microfluidic device further includes a column configured to separate the sample into non-fatty nucleated cell fractions.

According to some embodiments, the microfluidic device is configured to wash the non-adipocytes with a wash solution and substantially remove the enzyme in the dissociation fluid.

In accordance with one aspect of the present disclosure, a substantially isolated adipose tissue processing system is provided. The system includes a sample washing and dissociation chamber that washes the sample The polyester and dissociation chamber comprises three inlets, a first outlet and a second outlet, and a screen disposed between the three inlets and the first and second outlets; the block reduces the chamber, the block is reduced The chamber includes an inlet connector fluidly coupled to the first outlet of the sample wash and dissociation chamber, an outlet and a screen disposed between the inlet connector and the outlet; a reservoir for separating cells and other Debris removal, the reservoir having an inlet in fluid communication with the outlet of the bulk reduction chamber; and a waste collection chamber having an inlet in fluid communication with the second outlet of the sample wash and dissociation chamber; Microfluidic device.

According to some embodiments, the microfluidic device comprises a plurality of microfluidic channels having a substantially constant depth and at least one dimension less than 750 microns.

According to some embodiments, at least two of the plurality of microfluidic channels are disposed on one surface of the substrate.

According to some embodiments, the sample washing and dissociating chamber, the bulk reducing chamber, the reservoir, and the waste collection chamber are each included in the same sealed package.

The drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components illustrated in the various figures are represented by the same numerals. For the sake of clarity, not every component in each drawing can be labeled. All figures are to be regarded as illustrative unless otherwise indicated. In the drawings: FIG. 1A is a flow of a method according to an embodiment of the present disclosure Figure 1B is a flow diagram of a method in accordance with one embodiment of the present disclosure; Figure 2A is a schematic diagram of a sample processing apparatus in accordance with one embodiment of the present disclosure; and Figure 2B is an implementation in accordance with the present disclosure 2C is a schematic diagram of a flow control device according to an embodiment of the present disclosure; FIG. 2D is a schematic view of a sample processing device according to an embodiment of the present disclosure; 2A is a schematic diagram of a sample processing apparatus according to an embodiment of the present disclosure; and FIG. 2G is a sample processing apparatus according to an embodiment of the present disclosure; 3A is a front view of a sample processing apparatus according to an embodiment of the present disclosure; FIG. 3B is an isometric view of the sample processing apparatus of FIG. 3A; and FIG. 3C is a cavity of the apparatus of FIG. 3A An expanded view of a portion of the chamber; Figure 3D is an expanded view of a portion of the chamber of the apparatus of Figure 3A; and Figure 3E is a cross-sectional side view of a portion of the chamber of the apparatus of Figure 3A Figure 3F is a front elevational view of the sample processing device of Figure 3A including a clip selected as appropriate; Figure 4 is a front elevational view of the sample processing device in accordance with one embodiment of the present disclosure; A front view of a chamber of a sample processing device of one embodiment of the disclosure; FIG. 5B is a cross-sectional side view of a portion of the chamber of FIG. 5A; and FIG. 6A is a sample processing device according to an embodiment of the present disclosure Front view of the chamber; Figure 6B is a cross-sectional side view of the chamber of Figure 6A; Figure 7 is a cross-sectional side view of the chamber of the sample processing apparatus according to one embodiment of the present disclosure; A front view of a chamber of a sample processing device in accordance with an embodiment of the present disclosure; a view of FIG. 8B is an expanded view of the chamber of FIG. 8A; and FIG. 9A is a sample processing device according to an embodiment of the present disclosure. Front view of the chamber; Fig. 9B is an expanded view of the chamber of Fig. 9A; Fig. 10A is a front view of a portion of the sample processing apparatus according to an embodiment of the present disclosure; and Fig. 10B is a view of Fig. 10A Expansion of part of the sample processing device FIG.;. 11 photo shows a sample processing according to one embodiment of the present disclosure means 12 is a front view of a sample processing apparatus according to an embodiment of the present disclosure; FIG. 13A is a front view of a sample processing apparatus according to an embodiment of the present disclosure; 13A is a cross-sectional view of a portion of a chamber of the apparatus of FIG. 13A; and FIG. 14 is a front view of a sample processing apparatus according to an embodiment of the present disclosure; 15A is an illustration of a portion of a microfluidic device included in some embodiments of the present disclosure; 15B is a description of a microfluidic device included in some embodiments of the present disclosure; and FIG. 15C is a disclosure of the present disclosure Description of the microfluidic device included in some embodiments; 15D is an illustration of a microfluidic device included in some embodiments of the present disclosure; and FIG. 15E is a microfluidic device included in some embodiments of the present disclosure 16 is a front elevational view of a sample processing device in accordance with one embodiment of the present disclosure; and FIG. 17 is a microfluidic device included in some embodiments of the present disclosure BRIEF DESCRIPTION OF THE DRAWINGS Figure 18A is a photograph of a fluid treated in one embodiment of the present disclosure; and Figure 18B is a photograph of a fluid treated in one embodiment of the present disclosure.

The present disclosure does not limit its application to the construction and configuration details of the components described in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of various embodiments. Also, the idioms and terms used herein are for the purpose of description and should not be considered as limiting. The use of "including", "comprising", "having", "comprising", "involving" and variations thereof in this document is intended to cover the following items and their equivalents.

As used herein, the terms "sample", "tissue sample", "fat sample" and "fatty tissue" may include adipose tissue samples, such as lipoaspirate, and the terms are used interchangeably.

As used herein, the term "microfluidic device" may refer to a device having at least one fluid passage formed on a substantially one surface (which may be substantially flat or curved) and at least one lateral flow channel size less than about 1 mm. The lateral flow channel size can be, for example, the channel width or depth.

It has been found desirable to have a method for efficiently isolating related cells from adipose tissue and having a device that allows for easy and safe isolation of cells from adipose tissue. For research and clinical applications, it has been found that multiple treatments are required The actions of the adipose tissue samples are rationalized to minimize human error. In addition, it has been found to be important to treat adipose tissue samples in a substantially "isolated" environment in which a barrier is provided to isolate the fat sample from direct physical contact or fluid contact (eg, via unfiltered airflow) such that the external environment and/or Operators minimize or avoid the risk of contamination and infection. It has also been discovered that there is a need for a system and apparatus for adipose tissue processing in which many components and compartments are integrated into one piece to provide a substantially isolated environment to the sample. Systems and devices for adipose tissue processing are preferably easy to use, easy to manufacture, and have a low risk of failure. For many clinical and research applications, it may also be preferred that any portion of the device and system in direct contact with the adipose tissue sample is sterile and disposable.

Aspects and embodiments of the present disclosure provide a method of isolating certain constituent cell populations from adipose tissue samples. Other aspects and embodiments of the present disclosure provide an apparatus for achieving a method of isolating certain constituent cell populations from adipose tissue samples in an integrated, rationalized, safe, and easy to use manner.

Aspects and embodiments of the present disclosure provide an integrated device that includes a plurality of compartments for adipose tissue sample processing, which may include, but is not limited to, configured and configured for sample collection, Washing, stratification, mixing, heating, cooling, filtration, digestion, storage, fluid transfer and handling, cell labeling, sample processing, dissociation, waste collection, block removal, debris removal, cell concentration, cell enrichment, A compartment for cell isolation, cell culture, growth, culture, differentiation, amplification, and the like. The integrated device of an embodiment of the present disclosure may also include, for example, between control compartments The valve for the purpose of fluid flow. These devices can be adapted to integrate and rationalize a variety of adipose tissue sample processing actions, such as separating cells from adipose tissue, and can contribute to a variety of functions, such as enzymatic digestion, adipose tissue dissociation, washing, waste collection, debris removal, The cells are concentrated, labeled with an antibody, labeled with magnetic beads, expanded with a cell, and the like. These devices are particularly suitable for applications where safety, ease of use, and ease of manufacture are important. Some aspects and embodiments of the present disclosure include methods of using such a device.

One embodiment of the presently disclosed method for isolating cells from adipose tissue includes, but is not limited to, dissociating adipose tissue, releasing constituent cells, collecting released cells, and removing tissue debris. The method can further include an adipose tissue cleaning action prior to tissue dissociation. Adipose tissue cleaning actions can include removing or expelling undesired or excess fluid from the adipose tissue sample. Such undesirable fluids may include blood, body fluids, and tumescent solutions. The adipose tissue cleaning action can further include using a rinse solution to rinse or wash the adipose tissue. The method can further comprise the act of enriching or purifying the released cells. Additionally, the method can further include collecting waste liquid from the treatment. Another embodiment of the presently disclosed method for isolating cells from adipose tissue includes, but is not limited to, removal of excess fluid from adipose tissue, dissociation of adipose tissue and release of constituent cells, and removal of undesirable cells and debris. The method may further comprise one or more of: washing the adipose tissue sample, concentrating the relevant cells, washing the relevant cells, and performing immunological isolation using, for example, an antibody. In one embodiment, the concentration and/or wash associated cells can be performed using at least one microfluidic device. In another embodiment, one or Various actions can be performed by centrifugation. In yet another embodiment, one or more actions can be performed using hollow fibers.

In one embodiment of the present disclosure, a method of isolating a population of non-fat cells from adipose tissue is provided. Methods include, but are not limited to, removal of excess fluid from adipose tissue, washing of adipose tissue with a buffer solution, dissociation of adipose tissue using, for example, ultrasonic or enzyme-containing dissociation, removal of fat cells, free oil, matrix fibers, and tissue debris, Reduce red blood cells and thicken related cells. Cell enrichment can be achieved using a centrifuge, filter or microfluidic device. The method can further comprise one or more of the following: lymphopenia, cell washing, and immunological isolation. The action of removing excess fluid from adipose tissue, washing adipose tissue with a buffer solution, dissociating adipose tissue, removing fat cells, free oil, matrix fibers and tissue debris, and cell washing can be performed using, for example, sedimentation under gravity, centrifugation, and/or inclusion. The filter of the sieve filter is carried out.

A flow diagram of one embodiment of a method of separating a population of non-adipocyte cells from adipose tissue is shown in Figure 1A (generally indicated at 100). During the liposuction procedure, a tumescent fluid is often introduced into the patient to minimize blood loss, making the tissue firm and providing local anesthesia. The tumescent solution may contain 0.05% lidocaine and a concentration of adrenaline at a concentration of 1:1,000,000. The method includes the act of removing excess fluid from the fat aspirate adipose tissue (act 110). Excess fluid can contain blood and often contains a tumescent fluid that can interfere with downstream processing actions and associated cell separation. In one embodiment, because the density of the adipose tissue is lower than the excess fluid, the sample of the excess fluid removed can be layered into an adipose tissue layer and an excess fluid layer using gravity settling or centrifugation. The adipose tissue layer and excess fluid can then be separated into different containers to separate the adipose tissue from the excess fluid. A lotion containing, for example, a salt solution, a lactated Ringer's solution, a Hanks balanced salt solution, or a phosphate buffered saline solution can be applied to adipose tissue to wash tissue, and can be repeatedly divided. The layer is treated to wash the adipose tissue more thoroughly and remove excess fluid. In another embodiment, excess fluid can be drained using a filter that includes a screen. The wash solution can be added to the adipose tissue and drained using a filter to wash the tissue. This washing treatment can be repeated. In some embodiments, the filter can include a pore having a pore size of from about 30 micrometers ( μm ) to about 1 millimeter (mm), such as about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 μm. About 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm, or about 1 mm. In other embodiments, the filter can include a pore having a pore size of from about 70 μm to about 500 μm , such as about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm, or 500 μm. More specifically, the filter may comprise a mesh filter having a pore size of from about 70 μm to about 200 μm , such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170. Mm or about 200 μm. In another embodiment of the present disclosure, the filter pore size is smaller than the adipose tissue so that the filter traps the tissue. To effectively remove excess fluid, the washing action including adding a lotion and removing the lotion can be applied from about once to about ten times, such as once, twice, three times, four times, five times, six times, eight times or ten times. . The ratio between the volume of the fat sample and the volume of wash added to each wash may be between about 1:0.2 and about 1:10, such as about 1:0.2, about 1:0.3, about 1:0.5, about 1 : 0.7, about 1:1, about 1:2, about 1:3, about 1:5 or about 1:10. For example, 100 ml of lipoaspirate adipose tissue collected using tumescent liposuction can be mixed with 100 ml of lactated Ringer's solution and discharged using a nylon mesh having pores having a pore size of about 140 μm . This treatment can be performed three times to complete excess fluid removal. In another embodiment, each washing action comprises washing a volume between 0.6 and 4 times the sample volume (eg, 0.6, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, or 4 times the sample volume) The liquid is added to and removed from the sample, and the washing action is performed once, twice, three times or four times. In another embodiment, the excess fluid removal action may be combined with a stratified action of gravity followed by a filter discharge. In yet another embodiment, the wash solution can be added to the untreated fat tissue and mixed with the untreated fat tissue to dilute the excess fluid, followed by delamination or use a mesh filter to vent the fluid. Adding a lotion can make stratification or drainage more effective.

In another embodiment of the present disclosure, the excess fluid removal action can be performed by placing the sample in a container having an outlet and then draining the excess fluid without the use of a filter. In one embodiment, the container may further comprise a member for fluid control, such as a pinch valve. For excess fluid removal, excess fluid can be expelled through the outlet, and when the sample is near the outlet, the fluid control member can be used to close the outlet. The outlet may be smaller in size than the adipose tissue sample or may have a large size that allows the tissue sample to pass through. This action can be done manually or by means of a sensor (such as an optical sensor or an infrared sensor) that detects the sample relative to the exit. The wash solution can be added to the adipose tissue sample to wash or rinse the sample, and the excess fluid removal action can be repeated to clean the adipose tissue sample. Figure 1A The second action of the illustrated method (act 120) is used to dissociate adipose tissue. Adipose tissue can be dissociated using ultrasound. Adipose tissue can be dissociated using a dissociation solution. The dissociating solution may comprise an enzyme that breaks down the extracellular matrix of the adipose tissue. The dissociating solution may comprise collagenase, protease, proteinase, neutral protease, elastase, hyaluronidase, lipase, trypsin, release enzyme, DNase, deoxyribonuclease, pepsin or a mixture thereof. The dissociating solution can comprise a concentration between 0.1 mg/ml and 10 mg/ml (eg, about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.5 mg/ml, about 0.75 mg/ml, about 1 mg/ml, about 1.2 mg/ml, about 1.5 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 7 mg/ml or about 10 mg /ml) of collagenase. The dissociating solution may comprise trypsin. The dissociating solution may comprise collagenase and DNase. The dissociation fluid may comprise collagenase, hyaluronidase and deoxyribonuclease. The dissociation solution may comprise collagenase between 0.2 mg/ml and 5 mg/ml, hyaluronidase between 0.1 mg/ml and 4 mg/ml, and deoxyribonuclease between 1 unit and 400 units, each of which The unit is defined as the use of DNA as a substrate at 25 degrees C at pH 5.0 and a change in enzymatic activity of 0.001 per minute per minute for A260 at a concentration of 4.2 mM Mg++. The dissociation liquid may further comprise calcium ions and/or magnesium ions. The dissociating solution can include a concentration between 0.1 mM and 10 mM (eg, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 1 mM, about 1.5 mM, about 2 mM, about 3 mM). Magnesium or calcium ions of about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, or about 10 mM).

After adding the dissociation solution, it can be at a certain temperature (for example, about 37 degrees Celsius) The fat tissue is cultivated for a certain period of time, for example, from about 5 minutes to about 30 hours. During incubation, the adipose tissue and the dissociating liquid may be mixed intermittently and/or continuously to promote an effective reaction. The adipose tissue dissociation or incubation action can be carried out at 37 degrees Celsius for between about 10 minutes and about 120 minutes, such as about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes. Minutes, about 90 minutes, or about 120 minutes, wherein the adipose tissue samples are gently agitated continuously or intermittently in the dissociation fluid, wherein agitation occurs more frequently than every 3 minutes, such as every second, every 2 seconds, every 3 seconds, every 5 seconds, every 10 seconds, every 20 seconds, every 30 seconds, every 45 seconds, every 60 seconds, every 90 seconds, every 120 seconds or every 180 seconds.

At the end of the dissociation action, a metal ion chelating agent such as ethylenediaminetetraacetic acid (EDTA) may be added to sequester the metal ions and prevent enzymatic activity in the dissociation solution, and the temperature may be lowered to between about 4 degrees Celsius and 30 degrees Celsius. For example, room temperature, about 25 degrees Celsius, about 22 degrees Celsius, about 18 degrees Celsius, about 15 degrees Celsius, about 12 degrees Celsius, about 8 degrees Celsius, or about 4 degrees Celsius. After incubation, the temperature can be maintained at about room temperature, such as about 25 degrees Celsius, or between 18 degrees Celsius and 28 degrees Celsius. In another embodiment, after the dissociation action, plasma, platelet-enriched plasma or serum may be added to inhibit the enzyme in the dissociation solution.

The third action of the method of Figure 1A (act 130) is for removing free oil, matrix fibers, tissue fragments, and undesirable cells, such as fat cells. A range of apertures between about 10 μm and about 70 μm (eg, about 10 μm , about 15 μm , about 20 μm , about 25 μm , about 30 μm , about 35 μm , about 40) can be used. A mesh filter of pores of μ m, about 50 μm , about 60 μm or about 70 μm ) to substantially remove fat cells, matrix fibers, and tissue fragments. In another embodiment, the dissociated adipose tissue sample is passed through a pore size between about 20 μm and about 50 μm (eg, about 20 μm , about 22 μm , about 25 μm , about 30 μm , Screens of about 35 μm , about 40 μm and about 45 μm , and about 50 μm ). The filtrate passing through the sieve filter can be collected. Alternatively, fat cells, matrix fibers, and tissue fragments can be substantially removed using centrifugation.

The fourth, fifth, and sixth actions of the method of Figure 1A (acts 140, 150, and 160) include reducing red blood cells (RBC), concentration-related cells, and washing cells, respectively. The order of these three actions is interchangeable. For example, in one embodiment, the cells can be first concentrated, washed, and then reduced in RBC. In another embodiment, the cells can be washed first before they are concentrated. In yet another embodiment, the cells can be washed and concentrated simultaneously. The concentration action should be carried out in applications requiring a small volume. For example, cells isolated from adipose tissue are often cultured in the study. To obtain higher cell density in the cell culture flask and to increase culture efficiency, the cells can be concentrated. Isolated cells can also be used for transplantation or injection into humans or animals, where high cell concentrations are often required for better results. Cell concentration can also have the advantage of substantially removing the agents used in previous actions, such as enzymes in the dissociation solution, which inhibit cell growth or cause deleterious effects when transplanted into an animal or human patient. In one embodiment, a microfluidic device can be used to concentrate relevant cells and/or remove red blood cells. The microfluidic device can be configured to simultaneously achieve the fourth, fifth, and/or sixth actions. In addition, the microfluidic device can be configured to remove lymphocytes to reduce immunity The reaction is possible. In another embodiment, the concentration action can be achieved using centrifugation. In yet another embodiment, the concentration action can be achieved using a membrane filter. In yet another embodiment, the concentration action can be accomplished using hollow fibers (eg, hollow fiber membrane filters). In yet another embodiment, the red blood cell reduction action can be accomplished using a red blood cell dissolved solution (eg, ammonium chloride) that is selectively dissolved by the red blood cells.

The sixth action (act 160) of the embodiment shown in Figure 1A is for washing relevant cells and/or transferring relevant cells to the desired buffer. Centrifugation, buffer exchange and/or dialysis methods can be employed. Alternatively, the microfluidic device can also be configured to perform this action. This action further reduces the reagents used in the residual previous actions and may require this action when the relevant cells are used in a clinical transplant. In some embodiments, however, acts 140-160 may be omitted.

In one embodiment of the present disclosure, the method comprises pre-conditioning adipose tissue, dissociating adipose tissue, and refining the released cells. A flowchart of this embodiment of the method is shown in Figure 1B (generally indicated at 200). In one embodiment, the adipose tissue preconditioning action (act 210) includes draining waste liquid, removing waste liquid, removing excess fluid, flushing adipose tissue samples, washing adipose tissue samples, and/or breaking up adipose tissue samples. Shattering may be advantageous when the adipose tissue sample contains large pieces that are difficult to digest or dissociate with enzymes. The adipose tissue preconditioning action can be achieved using the aspects and examples of separating non-fat cell populations from adipose tissue as disclosed in the present disclosure. For example, the adipose tissue preconditioning action can include trapping the adipose tissue sample in the first container while allowing excess fluid, such as blood, a tumescent solution, or other bodily fluid, to pass into the second container. The intercepted adipose tissue sample can be achieved using a screen in the first container. In one embodiment, the mesh aperture is between about 70 μm and about 300 μm , such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, about 200 μm or about 300 μm. Alternatively, the cut-off adipose tissue sample can be achieved using a detector or sensor that detects the location of the adipose tissue sample relative to the first container without using a screen in the first container. The adipose tissue preconditioning action can further include adding and removing the irrigation fluid once or repeatedly to wash the adipose tissue sample. Tissue preconditioning can also be achieved using centrifugation in which adipose tissue samples are separated from excess fluid and/or waste liquid under centrifugal force. Alternatively, if the adipose tissue is in a condition that can be dissociated and refined, the adipose tissue preconditioning action can be omitted.

In one embodiment, the adipose tissue dissociation action (act 220) comprises incubating the adipose tissue sample at a temperature suitable for enzymatic digestion (eg, between about 32 degrees Celsius and about 38 degrees Celsius) in a dissociating solution comprising at least one enzyme. For a duration between about 3 minutes and 20 hours, the at least one enzyme is, for example, collagenase, protease, protease, neutral protease, elastase, hyaluronan, lipase, trypsin, papain, release enzyme, DNase, go Oxygen ribonuclease, pepsin or a combination thereof. In another embodiment, the adipose tissue dissociation action comprises passing the ultrasound through the adipose tissue sample. The cells are released from the adipose tissue sample during the adipose tissue dissociation action.

Purifying the released cells (action 230) may include cell concentration, cell washing, cell separation, cell isolation, Fragment removal, non-target cell removal, red blood cell reduction, or a combination thereof, is accomplished using filters, screens, hollow fibers, antibodies, microfluidic devices, or centrifuges. For many applications, such as care applications and field applications, it may be desirable to perform the entire method of the present disclosure in a short period of time (eg, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, or 120 minutes).

Many of the actions and examples disclosed herein for isolating non-adipocyte populations from adipose tissue can also be used in research learning and pharmacological testing systems. Separation of non-adipocytes can be performed for physiological, metabolic, and functional studies or for drug testing in pharmacological test systems. Many of the actions and embodiments disclosed herein can also be used in tissue engineering of transplants. Cells obtained using the methods and/or devices disclosed herein can be cultured in vitro to form tissue for in vitro culture for transplantation. Adipose-derived stem cells can be isolated to produce functional cells and tissues.

One embodiment of the present disclosure is a sample processing device schematically illustrated in FIG. 2A and generally indicated at 300. The sample processing device 300 includes a sample conditioning chamber 310 and a waste chamber 320. The sample conditioning chamber 310 includes a first inlet 305 for receiving a sample 340 (eg, a lipoaspirate or an adipose tissue sample from liposuction) for receiving a rinse solution (eg, a buffer solution, a saline solution, etc.) from the rinse fluid source 350. A second inlet 315, a first outlet 325 for collecting the sample 360 after the sample 360 is adjusted, and a second outlet 335 connected to the waste container 320. The second outlet 335 can include a member 345 that opens and closes a fluid connection between the conditioning chamber 310 and the waste container 320, such as a valve, pinch valve, piston, or the like. The sample conditioning chamber 310 causes the sample to drain its excess fluid into the waste container and wash it with the rinse liquid. The conditioning chamber may further include a sensor (preferably adjacent to the second outlet 335) to detect if the sample is near the outlet when excess fluid or irrigation fluid is drained into the waste container. The conditioning chamber can further include a filter disposed between the first inlet 305 and the second outlet 335 configured to trap adipose tissue during sample washing, sample rinsing, and removal of excess fluid. In some embodiments, the filter can include a filter comprising a pore size between about 30 μm and about 1 mm (eg, about 30 μm , about 50 μm , about 70 μm , about 85 μm , A pore of about 100 μm , about 120 μm , about 140 μm , about 200 μm , about 300 μm , about 500 μm , about 700 μm, or about 1 mm). In other embodiments, the filter can include a pore size of from about 70 μm to about 500 μm , such as about 70 μm , about 100 μm , about 140 μm , about 200 μm , about 300 μm, or 500 μ. The hole of m. The filter may comprise a pore size of from about 70 μm to about 200 μm , such as about 80 μm , about 90 μm , about 100 μm , about 120 μm , about 140 μm , about 170 μm or about 200 μ. m sieve filter. In another embodiment, the filter pore size is smaller than the adipose tissue piece such that the filter traps the tissue pieces.

The rinsing fluid can enter the conditioning chamber via a flow control device 330 (eg, a peristaltic pump) that controls the volume of rinsing fluid added to the conditioning chamber. The flow control device can include at least one valve and a variable volume container. For example, as schematically shown in FIG. 2B, the flow control device can include a piston 370 and a syringe 380. To dispense the measured volume, first turn the piston to the position where the inlet is fluidly connected to the syringe. Pull the syringe plunger to draw fluid from the inlet into the syringe. Then turn the piston to the outlet fluid Connect to the location of the syringe. Next, the syringe plunger is pushed to dispense fluid from the syringe via the outlet. Finally, the piston is again turned to fluidly connect the syringe to the inlet to complete a pumping cycle. The pumping cycle can be repeated until the desired volume of rinse is added to the conditioning chamber. As schematically shown in Fig. 2C, the flow control device may also include two check valves CV1, CV2 (also a valve that flows even in one direction) and a syringe. To dispense the measured volume, the syringe plunger is first pulled and then pushed to complete a pumping cycle. The first check valve (CV1) is configured to flow fluid from the inlet to the injector, and the second check valve (CV2) is configured to flow fluid from the injector to the outlet. Repeatable steps include pulling and pushing the piston to draw fluid into the syringe and out of the pump into the syringe for a desired period of time until the desired volume of rinse is added to the conditioning chamber. Alternatively, the syringe in the flow control device depicted in Figure 2B or Figure 2C can be replaced with a bag that can be inflated and deflated, or a container that can be controlled in a controlled manner.

Another embodiment of the present disclosure is a sample processing device schematically illustrated in Figure 2D and generally indicated at 400. The sample processing device 400 includes a sample dissociation chamber 410 and a cell refining device 420. The dissociation chamber includes a first inlet 405 that receives the sample, a second inlet 415 that receives the dissociation fluid from the dissociation source 430, and at least one outlet 425 that is fluidly coupled to the cell refining device. The dissociation chamber is configured to dissociate the sample into smaller compositions, such as single cells and small cell masses. The dissociation fluid can comprise an enzyme that decomposes the sample. For example, the dissociation fluid may comprise collagenase, protease, protease, neutral protease, elastase, hyaluronidase, lipase, trypsin, liberase, DNase, deoxyribonuclease, pepsin or mixtures thereof. The temperature can be controlled, for example, at about 37 degrees Celsius, and the sample in the dissociation chamber can be mixed with the fluid to aid in efficient enzymatic reaction and uniform dissociation. One embodiment of the dissociation chamber is a flexible bag. The bag may be repeatedly massaged, squeezed, rolled, shaken, shaken, partially squeezed and released, or otherwise agitated to aid in mixing. The dissociating liquid may also contain a cleaning agent such as Tween 20 or sodium lauryl sulfate. When ultrasound is used to dissociate the sample, the dissociation fluid can contain a medium that effectively conducts the ultrasound. The dissociation chamber can include a filter, such as a screen or filter. A filter can be used to maintain the sample in an effective dissociation position and/or to remove large fragments from the dissociated sample. In some embodiments, the filter can include a filter comprising a pore having a pore size of from about 30 μm to about 1 mm, such as about 30 μm, about 50 μm, about 70 μm, about 85 μm, about 100 Μm, about 120 μm, about 140 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm, or about 1 mm. In other embodiments, the filter can include a pore having a pore size of from about 70 μm to about 500 μm , such as about 70 μm, about 100 μm, about 140 μm, about 200 μm, about 300 μm, or 500 μm. The filter may comprise a mesh filter having a pore size of from about 70 μm to about 200 μm , such as about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm or about 200 μm. . In another embodiment, the filter pore size is smaller than the adipose tissue piece so that the filter traps the tissue.

The cell refining device is connected to the dissociation chamber. In some embodiments, the sample processing device further includes a valve 435 between the dissociation chamber and the cell refining device. The valve can be closed to allow incubation of the sample and the dissociation solution, and open to The released cells are allowed to enter the cell refining device.

The cell refining device is configured to receive the released cells from the dissociation chamber and to refine the released cells. In some embodiments, the cell refining device includes a chamber fluidly coupled to the dissociation chamber via an inlet 445, an outlet 455 for collecting refined cells 440, and a filter configured to remove large debris from the dissociated sample. The filter may comprise a filter having a pore size of between about 10 μm and about 100 μm , such as about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 50 μm, about 70 μm or about 100 μm. The filter may also include a screen having a pore size of between about 20 μm and about 60 μm , such as about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, About 50 μm and about 60 μm.

Another embodiment of the present disclosure is a sample processing apparatus schematically illustrated in FIG. 2E and generally indicated at 500. The sample processing device 500 includes a first chamber 510, a waste container 520, and a cell refining device 530. The cell refining device can include a mesh as disclosed above. The first chamber can act as a preconditioning chamber in which the sample can be washed prior to dissociation, and the dissociation chamber, wherein the sample can be dissociated into smaller compositions, such as individual cells or small aggregates of cells. The first chamber includes a first inlet 505 for receiving a sample and a second inlet 515 for receiving a rinse fluid from the rinse source 350. In some embodiments, the irrigation fluid and the dissociation fluid can enter the first chamber via the same inlet. In other embodiments, the first chamber includes a third inlet 525 for receiving the dissociation fluid from the dissociation source 430. The first chamber may further comprise a filter, such as a screen or filter, as described above with respect to the dissociation chamber and the conditioning chamber Said. The first chamber is fluidly connected to the waste chamber and the cell refining device. Valves V1 and V2, which may include, for example, pinch valves or pinch valves, may be used to control fluid flow. For example, during sample preconditioning, valve V1 can be opened to allow excess fluid and irrigation fluid to flow from the first chamber into the waste container. Both V1 and V2 can be turned off to allow incubation of the sample and the dissociation solution during sample dissociation. After dissociation, V2 can be opened to transfer the dissociated sample to the cell refining device.

In some embodiments of the present disclosure, the adipose tissue sample is pre-warmed to a temperature, such as 37 degrees Celsius, and then loaded into the first chamber 510. Preheating when processing adipose tissue samples can reduce the time required to process tissue samples. In other embodiments of the present disclosure, the adipose tissue sample is photoactivated using light having an average wavelength between 300 nm and 700 nm and subsequently loaded into the first chamber 510. Photoactivation increases the potency of cells in tissue samples. In yet another embodiment of the present disclosure, the adipose tissue sample is processed using sonic waves (i.e., sound waves that relax the tissue to make the tissue sample easier to dissociate) and subsequently loaded into the first chamber 510.

The sample processing device can further include a flow control device 330 for controlling the flow of irrigation liquid entering the first chamber as described above. Flow control device 330B, which may be similar to flow control device 330, may also be used to control the flow of the dissociation fluid injected into the first chamber. In some embodiments, the dissociation fluid is loaded into a syringe and subsequently injected into the first chamber.

It will be appreciated that the sample processing devices disclosed herein can have different variations and combinations. For example, as shown in FIG. 2F, an embodiment of a sample processing apparatus generally indicated at 500A can employ two valves (V1, V2). To control the flow of fluid between the first chamber, the waste container, and the cell refining device. The flow control device can include two check valves (CV1, CV2) and a volume measuring device 540, such as a syringe. The dissociation fluid can be connected to the first chamber via a check valve CV3.

Another embodiment of the sample processing apparatus of the present disclosure is schematically illustrated in Figure 2G (generally indicated at 500B) in which at least 3 pistons (SC1, SC2, SC3) are used to control fluid flow. For example, during sample preconditioning, excess fluid and irrigation fluid can be transferred from the first chamber to the waste container. The piston (SC3) can cut off the flow leaving the first chamber to allow incubation of the sample and the dissociation fluid during sample dissociation. After dissociation, the piston (SC3) can connect the first chamber to the cell refining device to transfer the dissociated sample to the cell refining device.

Figure 3A shows an embodiment of the apparatus schematically shown in Figure 2E. The device includes two plastic sheets joined together to form a plurality of chambers. The device can be used in a rational, easy to use, safe, and cost effective manner to facilitate the adipose tissue processing methods disclosed herein. The chamber 1 is a measuring chamber that can be inflated to a certain volume. The chamber includes an inlet (port 1) and an outlet (port 2). The chamber 1 can be designed to absorb fluid and dispense a predetermined volume of fluid, thereby controlling the volume of fluid to be dispensed into the chamber 2 via the inlet (port 3) for adipose tissue processing. Port 2 and port 3 may be fluidly connected via a tube, and the tube may be clamped to fluidly shut the port 2 and port 3 using a pinch valve, pinch valve, piston, check valve or other valve adjustment mechanism. To operate the chamber 1, the connection between the port 2 and the port 3 is initially closed. Fluid is introduced into the chamber 1 via port 1 to fully or partially inflate the chamber. Connect The port 1 is cut using, for example, a spring clip, a pinch valve, a piston, a check valve, or other valve adjustment mechanism. Next, the valve between the port 2 and the port 3 is opened to allow the fluid in the chamber 1 to flow into the chamber 2 when the chamber 1 is deflated. Venting of chamber 1 can be accomplished by externally squeezing and/or compressing the chamber by gravity, by siphoning, or by other methods known in the art. The chamber 1 can be positioned higher than the chamber 2 to facilitate the use of gravity to distribute fluid into the chamber 2. This treatment transfers the predicted amount of fluid into the chamber 2 as determined by the difference between the aerated volume and the deflated volume of the chamber 1. If a smaller volume of fluid is desired, the chamber 1 can be partially compressed, squeezed, and/or pinched to control the volume of fluid entering and/or exiting the chamber 1. Alternatively, chamber 1 may only be partially deflated to dispense a portion of the fluid therein. If a larger volume is desired, the aeration-deflation process can be repeated several times until the desired volume of fluid is transferred into the chamber 2.

Chamber 1, Port 1 and Port 2 may be embodiments of flow control devices schematically illustrated in Figure 2B or Figure 2C.

Port 1 and port 2 may include a check valve, also referred to as a one-way valve, which only allows fluid to enter and exit chamber 1 separately. The act of measuring and dispensing a set volume of fluid becomes extremely simple: depressurizing chamber 1 to allow fluid to enter and compress chamber 1 via port 1 to push fluid through port 2.

Alternatively, chamber 1 can be a storage chamber that provides the pre-packaged solution required for sample processing. For example, lactated Ringer's solution, balanced salt solution, saline solution, dissociation solution, lotion, rinse solution, solution containing ethylenediaminetetraacetic acid (EDTA) and/or enzyme solution may be packaged as part of the device. In the chamber 1.

In another embodiment, the measurement chamber can include a syringe that includes a piston that can draw and/or dispense a predetermined volume of fluid by pulling and pushing the piston. In yet another embodiment, the metrology chamber can include a flexible tube mounted to a peristaltic pump, wherein a peristaltic pump is used to control fluid flow.

The chamber 2 can be an embodiment of the first chamber 510 shown schematically in Figure 2E. The chamber 2 can be an adipose tissue washing chamber that includes one or more inlets and outlets. The chamber 2 can also serve as a dissociation chamber and can further comprise at least one screen, such as a filter mechanism comprising a screen, a semi-permeable membrane and/or a porous or microporous membrane. Adipose tissue samples such as lipoaspirate or fatty lipoaspirate tissue sheets can be introduced or loaded into the chamber 2 via the inlet (port 4). Excess fluid from the sample can be discharged through the screen through the connector 1 into the waste collection chamber (chamber 3). A wash solution can be added to the chamber 2 to wash and/or rinse the sample. The wash solution can be measured and dispensed into the chamber 2 via the chamber 1. A solution of the treated sample can be added to change the sample. A mixing member can be applied to the chamber 2 to make flushing and washing more efficient. For example, the chamber 2 can be massaged repeatedly, gently squeezed, shaken, shaken, partially squeezed and released, or otherwise agitated to aid mixing. The waste liquid can then be discharged into the waste collection chamber (chamber 3). The valve regulating member can be used to close the outlet of the chamber 2 (connectors 1 and 2) during mixing to allow thorough mixing between the washing liquid and the sample, followed by discharge of the waste liquid. Clips such as clip 1, clip 2 and/or clip 3 as illustrated in Figure 3F can be applied to grip the chamber and close the fluid connection between the chambers. The washing process can be repeated several times, for example 2, 3, 4 or 5 times. Adipose tissue samples can be trapped in the cavity during the washing action In room 2. For adipose tissue samples containing small pieces, the chamber 2 can include a mesh or membrane filter to effectively trap the sample. Multiple screens and/or filter layers (screen 1 and screen 2) may be combined to provide the desired adipose tissue retention.

After washing, the dissociation fluid can be added to the chamber 2 to dissociate the adipose tissue sample and release the cells. The chamber temperature can be set to an optimized temperature using a heating, cooling, and/or temperature control system to aid sample dissociation. For example, the device can be placed in an incubator, in a water bath, and/or in contact with a constant temperature plate to maintain the temperature at about 37 degrees Celsius for optimal enzymatic digestion. The dissociation fluid may comprise one or more enzymes to break down the connective matrix, the extracellular matrix, and the like. For example, collagenase can be used at 37 degrees Celsius to break down collagen fibers. Other reagents (including protease, protease, trypsin, proteinase K, dissociation enzyme, enzyme, lysate, hyaluronan, lipase, trypsin, liberase, DNase, DNase, pepsin or mixtures thereof) can also be used Digested in adipose tissue. The outlet of the dissociation chamber (chamber 2) (connector 1 and/or connector 2) can be closed during digestion. The chamber 2 can be repeatedly massaged, gently squeezed, shaken, shaken, partially squeezed and released, or otherwise agitated to aid in mixing and promote effective adipose tissue dissociation. When the digestive action is complete, the connector 2 can be opened to allow the released cells to exit the dissociation chamber. At least one screen in the chamber 2 can be used to remove or trap large pieces of debris. One or more additional washing actions including the addition of a wash solution may be applied to rinse out cells that may be captured in the chamber 2 after digestion.

The sample processing device shown in FIG. 3A may further include a debris removal chamber (chamber 4), which may include a mesh, a membrane filter, and/or another A sheet removal mechanism (eg, screen 3). The dissociated sample can be transferred to a debris removal chamber where large, unwanted cells (such as fat cells) and/or debris can be removed from the sample. The chamber 4 can also serve as a sample storage chamber in which the released cells are contained until further use. The released cells can be collected via port 5. The chamber 4 can be an embodiment of the cell refining device 530 shown schematically in Figure 2E.

Figures 3B and 3C show an embodiment in which a screen is incorporated into the chamber 4. The screen may be folded, sandwiched between two flexible sheets, and welded, glued, heat sealed or otherwise fused together to form the chamber 4. Similarly, a screen or membrane filter can be incorporated into the chamber 2. 3D and 3E respectively include an expanded view and a cross-sectional view showing one or more portions of the screen that can be used in one of the chambers 2 in the chamber 2. The screens included in chamber 2 and/or chamber 4 may include apertures that progressively become smaller along the flow path of fluid through chambers 2 and/or 4. For example, screen 1 (Fig. 3D) may have a nominal pore size of from about 100 μm to about 900 μm , and screen 2 may have a nominal pore size of from about 50 μm to about 200 μm , and the screen The nominal pore size of 3 (Fig. 3C) can range from about 10 μm to about 60 μm .

It should be understood that the present disclosure is not limited to the particular configuration of the embodiment shown in Figures 3A-3F. Embodiments of the present disclosure may include more or fewer chambers than those shown in Figures 3A-3F. Chambers with various functions are available and configured to achieve specific tasks including a specific sequence of actions. For example, the chamber can have functions including, but not limited to, sample collection, washing, rinsing, stratification, mixing, heating, cooling, filtration, digestion, storage, valve tuning, volumetric measurement, aspiration, Fluid transfer and operation, Cell labeling, sample processing, dissociation, waste collection, block removal, debris removal, dissolution, concentration, polymerase chain reaction (PCR), incubation, hybridization, cell culture, cell expansion, and the like.

Another embodiment of the sample processing device of the present disclosure is formed using two plastic sheets (generally illustrated by 600 in Figure 4). The chamber 1 is a sample washing and dissociation chamber comprising three inlets (port 1, port 2 and port 3), a screen (screen 1), and two outlet connectors (connector 1 and connector 2). The chamber 2 is a block reduction chamber that includes an inlet connector (connector 2), an outlet/inlet connector (connector 3), and a screen (screen 2). The chamber 3, optionally including the screen (screen 3), may be a reservoir for removal of separated cells and other debris, or a cell refining chamber. The chamber 4 is a waste collection chamber that includes an inlet tube (connector 1) in fluid communication with the outlet connector of the sample wash and dissociation chamber (chamber 1).

5A and 5B show another embodiment of a chamber 610 of the present disclosure that includes two screens. The two screens are configured so as not to substantially overlap. Each screen is folded and sandwiched between two flexible outer sheets. This embodiment may have the advantage of being easier to manufacture because only double-layer screens must be fused between the sheets.

6A and 6B show yet another embodiment of a chamber 620 of the present disclosure that includes at least one unfolded screen sandwiched between two flexible outer sheets. The screen is located between the inlet and the outlet and is configured such that fluid entering from the inlet must pass through the screen to reach the outlet. The inlet and outlet are on opposite sides of the screen. Since only one layer of screen must be sealed between the two sheets, this configuration has the advantage of being easy to manufacture.

Figure 7 shows yet another embodiment of a chamber 630 of the present disclosure that includes a pleated screen sandwiched between two flexible outer sheets. This configuration can have the advantage of increasing the screen area.

8A and 8B show yet another embodiment of the chamber 640 of the present disclosure wherein the screen is folded and sealed along the sealing edge to form a pouch which is then incorporated into the chamber. A piece of plastic screen (e.g., a polyamide screen) can be folded along the fold line and sealed along the two seal lines to form a screen pouch using, for example, heat sealing or high frequency welding. The mesh pouch can then be positioned between two flexible plastic sheets, such as polyvinyl chloride (PVC) sheets, and sealed along the sealing edge using, for example, heat sealing or high frequency welding to form a chamber.

9A and 9B show yet another embodiment of the chamber 650 of the present disclosure wherein the screen is folded, sandwiched between two flexible plastic sheets and sealed in the chamber. Herein, the chamber and screen are configured such that the sample entering the chamber via the inlet can exit through the outlet 1 without passing through the screen, while the portion of the sample exiting via the outlet 2 must pass through the screen. The folded line of the screen is approximately vertical. In another embodiment, the fold line can be at an angle relative to the vertical.

Any one or more of the screen configurations illustrated in Figures 5A-9B can be used in any of the chambers of any of the sample processing devices disclosed herein, such as chamber 2 or chamber 4 of sample processing device 500. One or more of and/or one or more of chamber 1, chamber 2 and/or chamber 3 of sample processing device 600.

10A and 10B show a connector between two chambers including at least one tube section, which may be included in the disclosure herein In any of the embodiments of the sample processing device. The plastic sheet is cut and traversed by the tube bridge from one chamber to the other. This configuration allows the valve adjustment member to access the tube. For example, a spring clip or slider clamp can be employed to open or close the fluid connection through the tube. The tube section may further comprise a soft and flexible section (tube 2 in Figures 10A and 10B) to aid in reliable tightening. This can be advantageous when a pinch valve is used. The pinch valve can be actuated manually, pneumatically or with a solenoid. The flexible tube can also help the peristaltic pump if necessary.

Embodiments of the sample processing device disclosed herein may further include other components, such as the components illustrated in Figure 11 (eg, a lancet that can be inserted into a lotion bag, one or more spring clips, a Y insertion site, a lancet, and/or Or a luer connector for connection to a syringe, and/or can be connected to other modules that are integral parts of a larger system. Spacers, injection ports, puncture needles, valves, check valves, tubes, adapters, Luer, female Luer locks, male Luer locks, syringes, pistons and/or other attachment mechanisms may be employed to enable this disclosure Portions of the embodiments are interconnected with other components.

Another embodiment of the present disclosure is a sample preparation device comprising a sample dissociation chamber (chamber 1), a waste container (chamber 2), and a cell refining chamber (chamber 3), which is generally in FIG. 700 instructions. The chamber 1 may optionally include a first screen to aid in sample washing, rinsing, and preconditioning. The screen comprises pores having a pore size between about 20 μm and about 600 μm . For processing a lipoaspirate sample, the mesh preferably has a pore size between about 40 μm and about 200 μm , for example about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, About 90 μm, about 100 μm, about 120 μm, about 140 μm, about 170 μm, or about 200 μm. a fluid connection of the piston control port 5, which can be closed during sample dissociation and incubation, which can be connected to the chamber 2 via the port 7 during sample washing, and which can be connected to the chamber 3 via the port 6 for collecting the released cells . The chamber 3 can include a second screen having a pore size between about 15 μm and about 150 μm . The second screen removes debris from the dissociated sample and refines the released cells, which can be collected at port 8. The device may further comprise an opening for receiving a sample, such as port 4, which may include a lancet port; an inlet for receiving irrigation fluid, such as port 1, which may include a lancet; and at least one inlet for receiving a dissociation fluid, such as port 2.

Yet another embodiment of the present disclosure is a sample preparation device comprising two sheets joined together in a predetermined pattern to form a sample dissociation chamber (chamber 1), a waste container (chamber 2), and a cell refining chamber (chamber 3) A flexible material, such as a plastic, is generally illustrated by 800 in Figure 13. The chamber 1 may include a first screen (screen 1) to aid in sample washing, rinsing, and preconditioning. The chamber 3 may include a second screen (screen 2) having a smaller aperture than the aperture of the first screen. The piston 1 controls the fluid connection between the chamber 1, the chamber 2 and the chamber 3. The chamber 1 includes an inlet (port 1) that includes a connector that can facilitate receipt of a sample from a syringe (eg, a syringe with a catheter tip). The chamber 1 further includes another port (port 2) connected to a piston manifold including the piston 2 and the piston 3. A rinse solution (eg, lactated Ringer's injection) can be coupled to the sample preparation device via a lancet. The syringe 1 together with the piston 2 acts as a flow control device that adds a defined amount of flushing fluid to the chamber 1. The dissociation fluid can be loaded into the syringe 2 and added to the sample preparation device via the piston 3. The dissociation fluid can be loaded into the syringe 2 in a concentrated form and reconstituted to a normal working concentration using the rinse solution. The chamber 3 includes an outlet, The released cells can be collected. In some embodiments, it is desirable to increase the pressure at the outlet. The chamber 3 can be hermetically sealed in the pressurized chamber (chamber 4). The chamber 4 includes a pressure port in which a pressurized fluid (e.g., compressed air) can be applied to indirectly pressurize fluid in the chamber 3 via the flexible sheet of the chamber 3. Figure 13B illustrates an embodiment of a chamber 3 and Figure 13C shows an embodiment of a pressurized chamber including a chamber 4. The chamber 3 is formed by joining together two flexible sheets at predetermined positions (Fig. 13B). A slit (cut 1) is created in the sheets to cause the other sheet to close the chamber 3 and form the chamber 4.

Another embodiment of the present disclosure includes a downstream processing unit (DPU 1000 illustrated in Figure 14) that can further process, refine, culture, amplify, and/or analyze the processed adipose tissue and/or isolated cells . The downstream processing unit can be configured to facilitate, for example, one or more of the following functions: sample washing, sample concentration, sample separation, sample enrichment, sample separation, buffer exchange, sample labeling, sample change, filtration, magnetic labeling, magnetic Separation, polymerase chain reaction (PCR), antibody interaction, affinity capture using antibodies, cell imaging, enzyme-linked immunosorbent assay (ELISA), protein preparation, protein purification, protein enrichment, mass spectrometry, high performance liquid chromatography , flow cytometry, cell sorting, functional analysis, cell culture, cell expansion, cell differentiation, immunophenotyping, cross-flow analysis, fluorescence in situ hybridization, deoxyribonucleic acid (DNA) hybridization, ribonucleic acid (RNA) Hybridization, deoxyribonucleic acid (DNA) reaction, ribonucleic acid (RNA) reaction, and the like.

The downstream processing unit can include a microfluidic unit that includes at least one microfluidic device. The microfluidic device can include at least one less than about 1 mm (eg, about 0.95 mm, about 800 μm , about 600 μm , about 500 μm , about 400 μm , about 300 μm , about 200 μm , about 150 μm) m, channel size of about 100 μm , about 80 μm , about 60 μm , about 50 μm , about 40 μm , about 30 μm , about 20 μm, and/or about 15 μm ). The microfluidic device can also include a channel having at least one substantially constant depth. For example, the microfluidic device can include channels having a depth of about 1 mm, about 800 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm. , about 80 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm or about 15 μm. The channel depth of the microfluidic device can be within 20% of the nominal channel depth. The microfluidic device can further comprise a channel substantially on one surface that can be substantially flat or curved. The microfluidic device can also include channels formed through one or more substantially planar surfaces.

Microfluidic devices can be formed using microfabrication, nanofabrication, and/or micromachining techniques including, but not limited to, photolithography, etching, reactive ion etching, deep reactive ion etching, wet Etching, stamping, injection molding, embossing, soft embossing, stereolithography, forming, soft lithography, anodic bonding, ultrasonic bonding, self-assembly, and/or other fabrication techniques known in the art.

Embodiments of the microfluidic unit of the present disclosure may include the devices disclosed in the following: International Application No. PCT/US10/061866, International Publication No. WO 2011/079217 A1, U.S. Patent No. 7,150,812 B2, U.S. Patent No. 7,735,652 No. US Pat. No. 8,021,614, U.S. Patent No. 8,186,913 B2, U.S. Patent Application Publication No. US 2012/0063664 A1, U.S. Patent Application Publication No. US 2011/0294187 A1, which is hereby incorporated by reference in its entirety in its entirety for all purposes in the the the the the ), inertial force, centrifugal force, deterministic lateral displacement, column array, column array, pinch flow, magnetic structure, antibody component, cell capture moiety, protein capture moiety, deoxyribonucleic acid (DNA) moiety, Ribonucleic acid (RNA) fraction, filtration, tangential flow filtration, ultrasonic focusing, compact flow, and the like.

It is to be noted that some embodiments of the present disclosure, in particular, embodiments of the microfluidic device disclosed in International Publication No. WO 2011/079217 A1 provide a device resistant to severe clogging and fouling, occlusion and Scale has so far been a serious problem that hinders the use of microfluidic devices for tangential flow filtration of digested adipose tissue.

Another embodiment of the present disclosure includes a hollow fiber unit that concentrates and/or washes the separated cells.

In another embodiment of the processing unit downstream of the microfluidic device, the microfluidic device washes the cells and removes undesired reagents. The downstream processing unit can include a buffer solution inlet to introduce a buffer solution to wash the cells. Cell washing can also be achieved using a microfluidic device designed to perform dialysis.

In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that reduces the enzyme concentration in the output of the downstream processing unit to less than about 1/10, for example, the enzyme concentration is reduced to about 1/10, about 1/20, about 1/30, about 1/40, about 1/50, about 1/70, about 1/100, About 1/150, about 1/200, about 1/400, about 1/500, about 1/750, about 1/1,000, about 1/2,000, about 1/5,000, about 1/10,000, about 1/20,000, About 1/50,000, about 1/100,000, about 1/200,000, about 1/500,000 or about 1/1,000,000. One embodiment of a microfluidic device that achieves such enzyme removal is disclosed in International Publication No. WO 2011/079217 A1, wherein the microfluidic device comprises a column and at least one buffer stream (e.g., a rinsing stream) is used to wash the cells.

In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that removes greater than 89% of the enzyme introduced during sample dissociation, such as removal of about 90%, about 95%, about 97% , about 98%, about 99%, about 99.5%, about 99.8%, about 99.9%, about 99.95%, about 99.98%, about 99.99%, about 99.995%, about 99.998%, about 99.999%, about 99.9995% or about 99.9999% of the enzyme introduced during sample dissociation.

In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that removes about 100% of the enzyme introduced during sample dissociation.

In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output having a concentration of collagenase: less than about 0.1 mg/ml, such as about 0.09 mg/ml, about 0.05 mg/ml, about 0.03 Mg/ml, about 0.02 mg/ml, about 0.01 mg/ml, about 0.007 mg/ml, about 0.005 mg/ml, about 0.003 mg/ml, about 0.002 mg/ml, about 0.001 mg/ml, about 0.0005 mg/ Ml, about 0.0002 mg/ml, about 0.0001 mg/ml, about 0.00005 mg/ml, about 0.00002 mg/ml, about 0.00001 mg/ml, about 0.000001 mg/ml, or about 0.0000001 mg/ml.

In yet another embodiment of the present disclosure, the downstream processing unit includes A microfluidic device that is substantially free of the output of the enzyme introduced during sample dissociation. In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output that is free of enzymes introduced during sample dissociation.

In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides substantially no output of collagenase introduced during sample dissociation. In yet another embodiment of the present disclosure, the downstream processing unit includes a microfluidic device that provides an output that is free of collagenase introduced during sample dissociation.

An example of a microfluidic device that can be used in any one or more of the sample processing devices disclosed herein is schematically illustrated in Figures 15A, 15B, 15C, 15D, and 15E, generally indicated at 910, 920, 930, 940, and 950, respectively.

Figure 15A shows a microfluidic channel 910 having a cell inlet, a buffer inlet, a cell outlet, and a buffer outlet. The width and/or depth of the microfluidic channel is so small, for example about 100 μm , that the cell sample and buffer form a stream of two laminar flows that are juxtaposed to each other without substantial convective mixing. The flow rate is configured to impart unwanted particles (e.g., enzymes) for a sufficient time to diffuse from the flow of cells to the flow of buffer stream. Because the cells are much larger than the unwanted particles, their diffusion is so small that they remain in the flow of cells. The cell stream and buffer stream exit the microfluidic channel via different outlets, thereby substantially removing unwanted particles.

Figure 15B shows another microfluidic channel 920 having a cell inlet, two buffer inlets, a cell outlet, and two buffer outlets. Channel and The flow rate is configured to diffuse unwanted particles from the cell stream to the buffer stream, thereby substantially removing unwanted particles. This configuration can have high removal efficiency because unwanted particles can be removed by two buffer streams.

For example, as illustrated in the microfluidic channel 930 illustrated in Figure 15C, the column or column can be positioned in the microfluidic channel to stabilize the cell and buffer flow, promote molecular diffusion, and/or support the microfluidic channel.

Microfluidic devices for dialysis can be configured in series to form a cascade. Figure 15D shows an example in which a buffer solution carrying unwanted particles is removed from buffer outlet 1 and fresh buffer solution can be introduced via buffer inlet 2 to substantially remove residual unwanted particles.

Figure 15E shows another embodiment of a microfluidic device 950 that includes a column array of channels and channels. The flow velocity and channel size of the fluid introduced into the channel are designed such that the fluid flow is layered. Typically, laminar and laminar fluid flows occur when the Reynolds number of the fluid in the microfluidic device is less than about one. The column array increases the effective diffusion coefficient of the particles in the buffer stream and improves the efficiency of removing unwanted particles (e.g., enzymes) from the cell stream.

In some embodiments, the buffer used to form the buffer stream in the microfluidic device is a rinse solution.

In yet another embodiment, the downstream processing unit includes a dialysis membrane.

Yet another embodiment of the downstream processing unit 1000 schematically illustrated in FIG. 16 includes a microfluidic device unit including at least one microfluidic device that concentrates the separated cells, and at least one collection reservoir, such as A bag is configured to receive the separated cells as an output of the microfluidic device. The downstream processing unit can further include a syringe coupled to the collection reservoir. After the sample has been processed using the microfluidic device, the output can be drawn into the syringe from the collection reservoir. The downstream processing unit may further comprise a waste reservoir, such as a waste bag.

In another embodiment of the present disclosure, the downstream processing unit includes a plurality of microfluidic devices to achieve a desired capacity, throughput, and function to process a large number of output samples.

Fluid transfer can use gravity, external pressure, vacuum, positive pressure, negative pressure, head height, pump (eg peristaltic pump), mechanism configured to squeeze the bag, squeeze the roll of the bag, squeeze the bag The board and/or other fluid transfer mechanisms known in the art are achieved. In one embodiment, the fluid can be transferred using a syringe. In another embodiment, as shown in Figure 13, the fluid can be transferred using an external air pressure applied to a chamber (e.g., a chamber 4 that encloses a bag containing cells (chamber 3)).

In another embodiment of the present disclosure, the downstream processing unit includes a cell culture chamber for culturing the cells. The cell culture chamber can be connected using a connector that allows the cell culture chamber to be detached. The cell culture chamber can be placed in an incubator where the temperature and conditions for cell growth can be optimized, for example at a temperature of about 37 degrees Celsius and a concentration of about 5% carbon dioxide. The cell culture chamber may further comprise an air permeable material, such as a filter membrane or a polyoxyxene rubber membrane, to allow gas exchange during cell culture.

One or more of the sample processing devices as disclosed herein may include a screen, a multi-layer screen, or a screen cascade (Fig. 5B). In some embodiments, the pore size of the screen for adipose tissue treatment (eg, the median or average size of the opening of the well) can be between about 1 μm and about 10 mm, such as about 1 μm, about 3 μm. , about 6 μm, about 10 μm, about 15 μm, about 25 μm, about 40 μm, about 70 μm, about 100 μm, about 140 μm, about 300 μm, about 700 μm, about 1 mm, about 2 mm or about 3 mm. The non-adipocytes are separated from the lipoaspirate tissue and the mesh aperture can be between about 10 μm and about 2 mm. In some embodiments, a mesh screen of about 40 μm , about 70 μm , about 100 μm , about 140 μm , about 250 μm, and/or about 700 μm may be employed, such as polyamide (nylon) ) Screen.

Another embodiment of the sample processing device of the present disclosure includes one or more chambers including two screens, the second screen being in fluid communication with the downstream of the first screen, wherein the pores of the second screen are substantially Less than the hole of the first screen.

Yet another embodiment of the sample processing device of the present disclosure includes one or more chambers including two membrane filters. The second membrane filter can be in fluid communication with the downstream of the first membrane filter. The pores of the second membrane filter may be substantially smaller than the pores of the first membrane filter.

Yet another embodiment of the sample processing device of the present disclosure includes one or more chambers including a track etch film filter.

Embodiments of the sample processing device or sub-assembly thereof as disclosed herein may be constructed using materials including, but not limited to, thermoplastic, acrylonitrile butadiene styrene (ABS), acrylic (PMMA), celluloid ( Celluloid), cellulose acetate, cycloolefin copolymer (COC), cycloolefin copolymer (COP), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), fluoroplastic (PTFE, and FEP, PFA, CTFE) , ECTFE, ETFE), Ionic polymer, liquid crystal polymer (LCP), polyoxymethylene (POM or acetal), polyacrylate (acrylic), polyacrylonitrile (PAN or acrylonitrile), polyamine (PA or nylon), poly Indole-purine imine (PAI), polyaryl ether ketone (PAEK or ketone), polybutadiene (PBD), polybutene (PB), polybutylene terephthalate (PBT), polyhexyl Lactone (PCL), polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), poly(trimethylene terephthalate) (PCT), polycarbonate (PC) Polyhydroxyalkanoate (PHA), polyketone, polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), polyether oxime (PES), chlorinated polyethylene (CPE), polyimine (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS) , polyphthalamide (PPA), polypropylene (PP), polystyrene (PS), polyfluorene (PSU), polytrimethylene terephthalate (PTT), polyurethane ( PU), polyvinyl acetate (PVA), polyvinyl chloride (PVC), polydichloroethylene (PVDC), styrene-acrylonitrile (SAN) and/or acrylonitrile butadiene styrene (ABS). For medical applications, flexible plastic sheets such as polyvinyl chloride (PVC), polyurethane (PU), ethylene vinyl acetate (EVA), polyamidamine (PA or nylon) can be used as tablets. material. In some embodiments, a sample processing device as disclosed herein can include two flexible sheets joined together and defining one or more chambers therein. In other embodiments, a sample processing device as disclosed herein can include a flexible sheet bonded to a rigid or semi-rigid material (eg, a thick plastic sheet and/or any one or more of the materials disclosed above) and in the flexible sheet and rigid One or more chambers are defined between the semi-rigid materials.

In some embodiments, the sheet thickness can be between about 0.1 mm and about 0.8. Between mm, for example about 0.1 mm, about 0.15 mm, about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, or about 0.8 mm. In other embodiments, the sheet thickness can be between about 0.2 mm and about 0.4 mm, such as about 0.2 mm, about 0.25 mm, about 0.3 mm, about 0.35 mm, or about 0.4 mm.

Materials for membrane filters and/or screens may include, but are not limited to, cellulose acetate (CA), glass microfibers (GMF), polyether oxime (PES), polypropylene (PP), regenerated cellulose (RC). Polyamide (PA or nylon), polytetrafluoroethylene (PTFE) and / or polyvinylidene fluoride (PVDF).

In some embodiments, the mesh material thickness can be between about 10 μm and about 1,000 μm , such as about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, About 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 175 μm, about 200 μm, about 250 μm, about 300 μm, about 400 Μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1 mm. In other embodiments, the mesh material thickness can be between about 50 μm and about 300 μm , such as about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, About 110 μm, about 125 μm, about 140 μm, about 160 μm, about 180 μm, about 200 μm, about 220 μm, about 250 μm, about 275 μm, or about 300 μm.

Embodiments of the present disclosure may be constructed using standard plastic manufacturing techniques including, but not limited to, plastic welding, heat sealing, injection molding, embossing, gluing, ultraviolet (UV) curing bonding, solvent bonding, hot air. Hot gas welding, freehand welding, speed contact welding (speed tip Welding), extrusion welding, contact welding, hot plate welding, high frequency welding, radio frequency welding, injection welding, ultrasonic welding, friction welding, spin welding, laser welding and/or solvent welding.

Embodiments of the present disclosure (e.g., sample processing devices schematically shown in any of Figures 2A-2G) can be constructed using high frequency welding of thermoplastic sheets. The welding die can be made of a metal such as aluminum, brass or stainless steel. The foldable screen member and the tube member are placed between the two thermoplastic sheets on the welding die. The thermoplastic sheet is then sandwiched using another welding die. A chamber is formed when pressure, temperature, and RF power are applied to the welding die. To seal a polyvinyl chloride (PVC) sheet having a polyamide screen, the following temperatures can be applied: between about 25 degrees Celsius and about 120 degrees Celsius, such as about 25 degrees Celsius, about 50 degrees Celsius, about 60 degrees Celsius, about 70 degrees Celsius, At about 80 degrees Celsius, about 90 degrees Celsius, about 100 degrees Celsius, about 110 degrees Celsius, or about 120 degrees Celsius, the following pressures are between about 10 psi and about 600 psi, such as about 10 psi, about 20 psi, about 30 psi, about 40 psi. About 50 psi, about 60 psi, about 80 psi, about 100 psi, about 150 psi, about 200 psi, about 300 psi, about 400 psi, about 500 psi, or about 600 psi, and the following RF power: at about 300 W Between 10 kW, for example about 300 W, about 500 W, about 600 W, about 700 W, about 800 W, about 900 W, about 1 kW, about 1.2 kW, about 1.5 kW, about 2 kW, about 2.5 kW. , about 3 kW, about 4 kW, about 5 kW, about 6 kW, about 7 kW, about 8 kW, about 9 kW, or about 10 kW. The RF power can be applied for a duration of between about 0.5 seconds and about 1 minute, such as about 0.5 seconds, about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 10 seconds, about 12 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, and about 60 seconds. The RF power can be applied several times at equal or different intensities to form a reliable seal. For medical applications, the device can be manufactured in a controlled clean environment and sterilized using standard sterilization techniques including, but not limited to, gamma radiation, ethylene oxide (EO) sterilization, and ultraviolet (UV) radiation. .

In some embodiments of the present disclosure, the sample preparation device is sterile. In some embodiments of the present disclosure, the sample preparation device is single use. Moreover, in some embodiments of the present disclosure, the sample preparation device is a substantial protective barrier that provides an isolation environment in which the protective sample is protected from direct physical contact or fluid contact (eg, via unfiltered airflow) such that the external environment and/or operation Personnel minimize or avoid the risk of contamination and infection.

It will be appreciated that embodiments of the present disclosure can act as a barrier to substantially reduce or eliminate any direct physical contact, fluid connection, and/or airflow connection between the adipose tissue sample and the surrounding environment. Any portion of the embodiments of the devices disclosed herein that may be in direct physical contact, fluid connection, and/or unfiltered gas flow connection to the sample may be sterile and/or single use. It will be appreciated that embodiments of the devices disclosed herein can substantially protect the sample from the risk of contamination and protect the operator from the risk of infection.

It will be appreciated that the present disclosure can be designed with an adipose tissue treatment device that is extremely easy to use. It should also be appreciated that the present disclosure greatly simplifies the manufacturing of such tissue processing devices and reduces the manufacturing costs of such adipose tissue processing devices. It should be further appreciated that embodiments of the present disclosure can be implemented using the same sample This device, which is substantially isolated from the surrounding laboratory or hospital environment, safely processes adipose tissue samples without substantial risk of contamination and infection.

Instance

Example 1. Isolation of non-adipocytes from human lipoaspirate tissue.

The human lipoaspirate is treated using a method including the actions depicted in Figure 1 and the devices as shown in Figures 3A and 11. The device is approximately 25 cm wide and approximately 40 cm long. The flexible plastic sheet is made of polyvinyl chloride (PVC) and the screen is made of polyamide (nylon). The nominal pore sizes of screens 1, 2 and 3 are about 140 μm , about 70 μm and about 35 μm, respectively . Approximately 40 ml of human lipoaspirate was collected from the consent donor using tumescent liposuction and treated within 24 hours from collection. Samples were shipped and stored at 4 degrees Celsius prior to treatment.

Initially, clips 1 and 2 are applied to close connectors 1 and 2 (Fig. 3F). Approximately 100 ml of lipoaspirate was added to the device via a lancet (Fig. 11). The clip 2 is opened to discharge excess fluid containing blood and a tumescent solution to the chamber 3 under gravity. After the excess fluid is substantially removed, the clip 2 is closed and the spring clip 1 is opened to allow about 50 ml of lactated Ringer's solution to enter the measurement chamber (chamber 1). Close the spring clip 1 and open the spring clip 2. The chamber 1 was squeezed using two plates to transfer the lactated Ringer's solution to the chamber 2. This fluid measurement and transfer process was repeated until approximately 100 ml of lactated Ringer's solution was added to chamber 2. A mild massage of chamber 2 was used to mix the lactate Ringer's solution with the lipoaspirate sample to wash the sample. The clip 2 is opened to discharge the waste liquid. This washing action was performed three times in this paper.

The tissue dissolution line is used to close the clips 1 and 2 and to add the dissociation liquid from the Y insertion site to the chamber 1 (Fig. 11). The dissociation solution contained 200 mg of collagenase and 50 mg of DNase I dissolved in 20 ml of lactated Ringer's solution. More lactated Ringer's solution was introduced into chamber 1 to dilute the dissociation solution. The dissociation liquid is then transferred to the chamber 2. The lactated Ringer's solution is again introduced into the chamber 1 to wash the chamber 1 and transfer the residual dissociation to the chamber 2. About 100 ml of lactated Ringer's solution was added to the chamber 2 during the tissue dissolution action. The device was placed in a 37 degree Celsius incubator and massaged frequently to effectively mix the tissue sample with the dissociation fluid. The tissue dissolution action takes about 45 minutes to 60 minutes.

After dissolution, the clip 1 is opened to allow the released cells to enter the debris removal chamber (chamber 4 in Figure 3A). The clip 2 is then opened to pass the sample through the screen 3. Tissue debris and fat cells are substantially removed during this action.

The solution of the effluent chamber 4 is then fed under gravity into the microfluidic device 1100, as illustrated in Figure 17 and in the International Application No. PCT/US10/061866, which is incorporated herein by reference for all purposes. Revealed. The microfluidic device includes about 110 modules disposed on the substrate plane. Each module includes approximately 900 columns configured in four columns. The depth of the microfluidic channel is about 30 μm .

The output of the microfluidic device is shown in Figures 18A and 18B. Concentrated non-fat cells are collected essentially in the nucleated cell product fraction (Fig. 18A). The volume of the nucleated cell product fraction is reduced to about 1/8 compared to the input. Then stain the cells with acridine orange (2 mg/l) and use fluorescent light. Micromirrors are used for imaging. The image shows that the fat cells are substantially absent from the product output of the microfluidic device, and the non-fatty nucleated cells are substantially collected in the nucleated cell portion. The images also show that the cells are in good shape and the product output is essentially free of disrupted cells.

The ADAM MC automated mammalian cell counter was used to further characterize total nucleated cell counts and adherent surviving nucleated cell counts of cells isolated using the disclosed methods and devices. The output chamber 4 having a total of about 6.0 × 10 ⁵ th fat aspirates nucleated cells / ml of the treated. Alpha-MEM supplemented with 10% fetal bovine serum (FBS) was used as a medium for adhering to viable nucleated cell counts. The output of chamber 4 is sampled and cultured in culture medium (e.g., cell culture dish or cell culture) for 3 days at 37 degrees Celsius in culture medium. After 3 days, the medium was discarded and the cell culture chamber was washed with Dulbecco's phosphate buffered saline solution. The cells adhering to the culture chamber were then released from the chamber surface for 3 minutes using trypsin. The adherent surviving nucleated cell count was about 1.5 x 10 ⁵ /ml of processed lipoaspirate.

Example 2. Method of isolating non-fat cells from human lipoaspirate tissue using a sample processing bag device.

Human lipoaspirate was treated using a sample processing bag device as depicted in Figure 13. The apparatus includes a sample dissociation chamber (chamber 1), a waste chamber (chamber 2), and a cell refining chamber (chamber 3). The device is approximately 24 cm wide, approximately 36 cm long and is made of two sheets of polyvinyl chloride (PVC) each approximately 0.3 mm thick. The sample dissociation chamber and the cell refining chamber include a first nylon mesh (mesh 1) and a second nylon mesh (screen 2). The first screen has a pore size of about 125 μm and the second screen has a pore size of about 25 μm . The aperture of the first screen may alternatively be between about 70 μm and about 160 μm , such as about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm. , about 140 μm, about 150 μm or about 160 μm. The aperture of the second screen may alternatively be between about 20 μm and about 50 μm , such as about 20 μm, about 22 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, or about 50 μm. .

Human lipoaspirate was collected from consented donors using tumescent liposuction and treated within 6 hours from collection. Samples were shipped and stored at 4 degrees Celsius prior to treatment.

Initially, the piston 1 is set to a position connecting the chamber 1 and the chamber 2. A dissociation solution containing 100 mg of collagenase, 100 mg of hyaluronidase, and 20,000 U of deoxyribonuclease was loaded into the syringe 2. The sample processing bag device was attached to the rinse bag containing lactated Ringer's injection using a needle.

Approximately 75 ml of the lipoaspirate sample was injected from chamber 1 into chamber 1 using a syringe with a catheter tip.

A washing action is applied to clean the liposuction sample. In order to start the washing cycle, the piston 1 is set to a closed position in which the chamber 1 is fluidly cut from the chambers 2 and 3. About 100 ml of lactated Ringer's injection was injected into the chamber 1 using the syringe 1 and the piston 2 as a flow control device, and the syringe 1 and the piston 2 were operated using the following sequence of actions: (a) the adapter piston 2 was used to make the rinse liquid Connected to the syringe 1; (b) draw the flushing fluid into the syringe 1; (c) transfer the piston 2 to connect the syringe 1 to the chamber 1; (d) flush the liquid The syringe 1 is injected into the chamber 1. This sequence can be repeated until the desired volume of solution is added to the dissociation chamber.

The chamber 1 is then massaged to mix the rinse with the sample, and the piston 1 is switched to discharge excess fluid (i.e., waste) into the chamber 2. After the discharge, the first wash cycle is completed. Repeat the wash cycle twice.

The washing action can include one or more wash cycles, such as one, two, three, four or five cycles.

After washing, the dissociation liquid is added to the chamber 1. About 100 ml of rinsing liquid was also added to the chamber 1. The sample processing bag device was then incubated at 37 degrees Celsius for a incubation time of about 60 minutes. The chamber 1 was massaged during this time to mix the sample with the dissociation solution. In some embodiments, other incubation times may also be used, such as about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 75 minutes, about 90 minutes, or about 120 minutes.

After incubation, individual cells released from the sample are passed through the piston 1 into the chamber 3 while large tissue fragments are captured by the screen 1 and remain in the chamber 1. The screen 2 in the chamber 3 further removes large fat cells and debris from the dissociated sample. Non-fat cells (including ectodermal cells, adipose-derived stem cells, and progenitor cells) are then collected at the outlet of the chamber 3.

To concentrate the released cells and the removed residual red blood cells and debris, the released cells collected at the outlet of the chamber 3 of the sample processing bag device are then passed through the first as disclosed in International Publication No. WO 2011/079217 A1. Microfluidic device. The microfluidic device includes 73 modules disposed on the surface of the substrate. Each module includes approximately 1,300 columns configured in four columns. The microfluidic device comprises channels at substantially the same depth, between about 35 μm and about 50 μm . In another embodiment, the microfluidic device can include a channel having a depth of between about 30 μm and about 80 μm , such as about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm or about 80 μm. The cells output from the microfluidic device were concentrated approximately three times. In another embodiment of the invention, the microfluidic device can be used to concentrate cells greater than about 2.5 times, such as about 3 times, about 4 times, about 5 times, about 6 times, about 8 times, about 10 times, about 12 times. Multiplier, about 15 times, about 20 times, about 25 times, about 30 times, about 40 times, about 50 times, about 60 times, about 80 times, about 100 times or about 125 times.

To remove the enzyme, the cells are treated via a second microfluidic device as disclosed in International Publication WO 2011/079217 A1. The second microfluidic device includes 83 modules disposed on the surface of the substrate. Each module includes approximately 900 columns configured in four columns. The rinsing fluid stream is introduced into each module and the cells are transferred to the rinsing fluid stream and separated from the enzyme.

Enzyme-linked immunosorbent assay (ELISA) was applied to measure the residual amount of collagenase after the second microfluidic device. The results showed that the collagenase concentration was reduced to 1/1,000, and the purified cells contained less than 0.001 mg/ml collagenase.

Having thus described a number of aspects of the at least one embodiment, it should be understood that various changes, modifications, combinations and improvements are readily apparent to those skilled in the art. Such changes, modifications, combinations and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the disclosure. Accordingly, the foregoing description and drawings are by way of example only.

Claims (26)

An apparatus for separating non-fat cells from an adipose tissue sample, the apparatus comprising: a first sheet of material; a second sheet of material bonded to the first sheet of material; and a plurality of chambers, the plurality of a chamber is defined between the first piece of material and the second piece of material, the plurality of chambers comprising: a sample dissociation chamber comprising an inlet and an outlet; a waste collection chamber, the waste collection chamber including a An inlet in fluid communication with the outlet of the sample dissociation chamber; and a cell refining chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet. The apparatus of claim 1, wherein the sample dissociation chamber further comprises a mesh filter comprising pores having a pore size between 70 μm and 300 μm. The apparatus of claim 1, further comprising a screen filter included in the cell refining chamber and comprising a pore having a pore size between 20 μm and 50 μm. Such as the equipment of claim 1 of the patent scope, wherein the sample dissociation room Further comprising a first screen filter, the first screen filter comprising a hole having a first aperture, and wherein the cell refining chamber further comprises a second screen filter, the second screen filter comprising a second a hole of the aperture, wherein the second aperture is smaller than the first aperture. The apparatus of claim 1, further comprising a member that controls a fluid connection between the sample dissociation chamber, the waste collection chamber, and the cell refining chamber. The apparatus of claim 5, wherein the member that controls the fluid connection comprises a piston. The apparatus of claim 1, further comprising a flow control device configured to introduce at least one of a rinse solution and a dissociation liquid into the sample dissociation chamber and having a fluid with the sample dissociation chamber Connected to the exit. The apparatus of claim 1, further comprising a member that applies pressure to the sample dissociation chamber and one of the cell refining chambers. The apparatus of claim 1, further comprising a downstream processing device in fluid communication with the outlet of the cell refining chamber and comprising at least one microfluidic device, the at least one a microfluidic device configured to separate a fluid output from the cell refining chamber into a first solution and a second solution, the first solution having one or more associated cells at a first concentration and the second solution having less than the first The one or more related cells at a concentration of the solution, wherein the related cells comprise non-fat cells isolated from the adipose tissue sample. A sterile and substantially isolated adipose tissue treatment system, the tissue treatment system comprising: a tissue processing chamber including an inlet, an outlet, and at least one screen filter, the at least one screen filter being disposed Between the inlet of the tissue processing chamber and the outlet of the tissue processing chamber; a waste collection chamber, the waste collection chamber and the tissue processing chamber being included in the same housing, the waste collection chamber including an outlet with the tissue processing chamber a fluid communication inlet; and one of: a debris removal chamber including a debris removal mechanism; and a sample collection chamber, the sample collection chamber and the tissue processing chamber being included in the same housing and being treated with the tissue The chamber is in fluid communication. A method of treating a fatty tissue sample in a tissue processing system, the method comprising: introducing a sample of adipose tissue to be treated via an inlet of the first chamber Processing the adipose tissue sample in the first chamber; and displacing cells from the first chamber through the outlet of the first chamber via the same housing as the first chamber The inlet of the second chamber is transferred into the second chamber. The method of claim 11, wherein processing the adipose tissue sample comprises dissociating the adipose tissue sample. The method of claim 11, wherein processing the adipose tissue sample comprises removing excess fluid from the adipose tissue sample in the first chamber. The method of claim 11, wherein treating the adipose tissue sample comprises washing the adipose tissue sample with a rinse solution in the first chamber. The method of claim 11, wherein treating the adipose tissue sample comprises washing the adipose tissue sample with a rinsing liquid in the first chamber, and dissociating in the first chamber using a dissociating solution comprising at least one enzyme The adipose tissue sample. The method of claim 15, wherein the dissociating liquid comprises collagenase. The method of claim 15, wherein the dissociating liquid comprises collagenase, deoxyribonuclease, and hyaluronidase. The method of claim 15, wherein dissociating the adipose tissue sample using the dissociation solution occurs at about 37 degrees Celsius. The method of claim 11, further comprising using a mesh filter to remove debris, the mesh filter being included in the second chamber. The method of claim 19, wherein the mesh filter has a pore size between 15 microns and 100 microns. The method of claim 11, further comprising: trapping the sample in the first chamber and passing the waste liquid through a screen filter included in the first chamber and the first chamber The outlet is transferred into the third chamber via an inlet of a third chamber that is included in the same housing as the first chamber. The method of claim 11, further comprising enriching the non-fat cell population using a microfluidic device. Such as the method of claim 22, wherein the non-fat The cells contain stem cells. The method of claim 11, further comprising collecting cells from the tissue processing system. The method of claim 11, further comprising processing the cells in a downstream processing device, the downstream processing device being in fluid communication with the outlet of the second chamber and comprising at least one configured to separate the cells A microfluidic device that is a first solution and a second solution, the first solution having a first concentration of non-fat cells and the second solution having a concentration less than the concentration of the first solution. The method of claim 24, wherein the collected cells are stromal blood vessel partial cells.