Patent Application
20220001091
A portion of the disclosure of this patent document contains materials that are subject to copyright protection. The copyright owner has no objection to any reproduction by anyone of the patent disclosure, as it appears in the United States Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
The present invention relates to isolation and purification of biological materials, and more particularly to a system introducing a stopcock valve for liquid component fractionation and application methods thereof. More specifically, the fractionation system can be employed for isolation and purification of Platelet-Rich Plasma (PRP) from whole blood, fractionation of fat components, lipids fractionation, and other fractionation situations, while there are differences in relative weights among the fractions in a liquid solution or liquid mixture.
PRP (Platelet-Rich Plasma) works by delivering a supra-physiologic amount of growth factors and cytokines contained within the platelet granules. The many growth factors in the platelet granules include TGF-β1, PDGF, β-FGF, VEGF, EGF, IGF-1, etc., which explains why PRP could be used for many clinical indications (De Pascale M R, Sommese L, Casamassimi A, Napoli C. Platelet derivatives in regenerative medicine: an update. Transfus Med Rev. 2015;29:52-61). In orthopedic medicine, PRP injection therapy is promising with clear evidence of efficacy and safety, based on the fact that PRP injections are being used to treat torn tendons, tendinitis, muscle injuries, arthritis, and joint injuries. For chronic wounds, PRP concentrates applied on the wound surface could provide accelerated healing. PRP injection is also being worked on for injecting into the scalp for hair regrowth, and encouraging data are being presented in many publications (Gentile P, Garcovich S, Bielli A, Scioli M G, Orlandi A, and Cervelli V. The Effect of Platelet-Rich Plasma in Hair Regrowth: A Randomized Placebo-Controlled Trial Stem Cells. Transl Med. 2015 November; 4(11): 1317-1323). Overall, the usefulness of the various growth factors contained in PRP makes it a very useful tool in the field of orthopedics, general surgery, plastic surgery, aesthetic medicine and dermatology, and etc.
The normal human platelet count ranges anywhere from 120,000 to 450,000/μL of blood. Because platelets exist in an inactive, suspension form in the blood, the isolation of them from the other components of the blood is preferably carried out by physical means, such as centrifugation, in order to avoid accidentally activating platelets prematurely.
In general, platelets could be isolated by two different approaches after the blood is mixed with an anticoagulant:
(1). Centrifugation at low g force, such as 100 g (“soft spin”). At these settings, the platelets remain suspended in the plasma. The supernatant is concentrated two folds in platelet numbers because red blood cells (RBC) account for about 45% of the total volume. This PRP supernatant is removed from the condensed red cells, then centrifuged at a higher g force (hard spin) to pellet the platelets from the plasma.
(2). Centrifugation at a high g force (“hard spin”), such as 500 g and above, to begin with. This causes the platelets to become separated from the plasma. Because platelets are lighter than leukocytes and red cells, with red cells being the heaviest, they are suspended in the middle-placed “buffy coat” layer, which includes the platelets and the white blood cells, as show in
PRP preparations are typically further categorized into leukocyte-rich PRP (LR-PRP) preparations, defined as having a neutrophil concentration above baseline, and leukocyte-poor PRP (LP-PRP) preparations, characterized by having a leukocyte (neutrophil) concentration below baseline (Le A D K, Enweze L, DeBaun M R, and Dragoo J L. Current Clinical Recommendations for Use of Platelet-Rich Plasma. Curr Rev Musculoskelet Med. 2018 December; 11(4): 624-634). They are found to have different clinical indications for orthopedic medicine.
There is no consensus on the optimal PRP preparation with respect to the concentrations of specific blood components. Currently, many different commercial PRP harvesting systems are available on the market. As such, variation exists in the PRP collection protocols and preparation characteristics depending on the individual commercial system, giving each PRP system unique properties. The commercial systems often differ in their platelet capture efficiency, isolation method (one- or two-step centrifugation), the speed of centrifugation, and the type of collection tube system and the operation (Le A D K, Enweze L, DeBaun M R, and Dragoo J L. Current Clinical Recommendations for Use of Platelet-Rich Plasma. Curr Rev Musculoskelet Med. 2018 December; 11(4): 624-634) (Degen R M, Bernard J A, Oliver K S, Dines J S. Commercial separation systems designed for preparation of platelet-rich plasma yield differences in cellular composition. HSS J. 2017;13:75-80) (Magalon J, Bausset O, Serratrice N, Giraudo L, Aboudou H, Veran J, Magalon G, Dignat-Georges F, Sabatier F. Characterization and comparison of 5 platelet-rich plasma preparations in a single-donor model. Arthroscopy. 2014; 30:629-638). Generally, fresh whole blood is drawn and mixed with an anticoagulant. Next, centrifugation(s) separates red blood cells (RBCs) from platelet-poor plasma (PPP) and the “buffy coat,” which contains concentrated platelets and leukocytes, as shown in
However, in addition to some patient-specific factors such as people to people variations, health states or medications taken, different commercial systems influence both the quality and quantity of the harvested PRP, creating challenges in the downstream applications (Le A D K, Enweze L, DeBaun M R, and Dragoo J L. Current Clinical Recommendations for Use of Platelet-Rich Plasma. Curr Rev Musculoskelet Med. 2018 December; 11(4): 624-634). Some of the main issues with the current available commercial kits include one or more of the following: long preparation time, cumbersome to use, low harvest rate, too much contamination by red blood cells, lack of ability to concentrate platelets beyond two folds, lack of ability to differentially isolate LR-PRP and LP-PRP, disturbance of the “buffy coat” during the collection stage, inconsistent results, and not to mention that many require the purchase of a separate special centrifuge, other than a regular, easily available benchtop centrifuge.
Meanwhile, in the field of fat fractionation, the main issue would be to increase the efficiency of separating oil from the fat component. The fat could be a preparation of regular fat, nano fat, or stromal vascular fraction (SVF) which harbors the fat stem cells.
Typically, surgeons would use either the conventional sedimentation method or a simple centrifugation method for fat graft purification. However, the sedimentation method, letting the lipoaspirate sit for 10 to 30 minutes, does not allow removal of oil, and the amount of water and RBC sedimented to the bottom is not consistent. Not to mention that in some fat harvests, the oil content could be anywhere from 5 to 80%, making the results of fat grafting unpredictable. Because of this and the fact that free lipids might harm the survival of fat grafts, it is better to use centrifugation to fractionate the fat allowing oil to be removed. However, what troubles the surgeons about the centrifugation method is the limited ability of removing the extremely sticky oil from the top of a tube. Even though there are quite a few commercial systems available for removing oil, most of these are either too expensive, or hard to use. And some do not even work as claimed.
As far as nano fat is concerned, the situation is even more complicated. Nano fat is created by first shuffling the harvested fat 30 times through a small “holed” (1.2 mm) Tulip LuerTransfer® (Tulip Medical Products, San Diego, Calif.) and then, subjecting the resulting fat to be pressed through a 400 micron screen. The recommendation is to use the emulsified product directly, or as is. However, this practice may not be sound considering what the components are after a centrifugation process, as shown in
Autologous fat stromal vascular fraction (SVF) contains most of the stromal stem cells. It could be prepared mechanically by ultrasonic sonication, homogenization, fine trituration, mortar and pestle treatment, etc. Traditionally, it has always been a difficult task to harvest SVF in a clean way. While the number of SVF cells are in the numbers of 105 to 106 per ml of fat, the single cell numbers in the finely homogenized fractions in suspension are in the ranks of 109. Even a diminutive, almost ignorable amount of contamination of the SVF by the above-mentioned fractions would drastically undermine the validity of the SVF cell counts at the end of the purification, and the subsequent treatment regimens or related experiments. This kind of contamination takes place very easily and very frequently with most, if not all, of the systems currently in use, for reasons such as the contaminating fractions sticking to the walls of the tube/bottle, or some almost invisible small pieces breaking away from the main fraction layer during the SVF collection. These are further complicated by the fact that the morphology of pre-adipocytes and small fat cells are not much different from the SVF cells when in suspension. Considering these, it is no wonder that the numbers of harvested “stem cell” from fat vary from 104 to 2.2×107 (WO2015005871A1, WO2014000031A1, and U.S. Pat. No. 8,440,440B2) among different publications. And this fact significantly hampers the progress in the field of fat stem cell research and clinical applications. As far as we know, currently, there is no reliable method or commercially available system for the purpose of purifying SVF without any risk of contamination by the other fractions.
Autologous fat is harvested routinely, i.e., with suction-assisted lipectomy or liposuction. Since the water content in a fat preparation varies depending on the amount of tumescent fluid used, the amount of time waited, and the inter-personal differences, most surgeons prefer to centrifuge down the fat to remove the free oil and water content, in order to perform fat grafting or other procedures in a more predictable way. The centrifugation force could be anywhere from a few g to 10,000 g, although at higher g forces, the survival of the fat grafts may not be as good (Strong A L, Cederna P S, Rubin J P, Coleman S R, and Levi B. The Current State of Fat Grafting: A Review of Harvesting, Processing, and Injection Techniques. Plast Reconstr Surg. 2015 October; 136(4): 897-912). The centrifugation could be 5 to 10 minutes. The usual speed to be used is around 500 g.
However, it is not an easy task to separate out the desired fat fraction after the fat is centrifuged, as shown in
Quite a few commercial systems (Salinas H M, Fernandes J R, Westman A M, Colwell A S, Broelsch G F, McCormack M C, Randolph M A, and Austen W G. Comparative analysis of processing methods in fat grafting. Plast Recon Surg., 2014 June; 134(4):1097) are available, but most do not provide satisfactory results in terms of separating oil from fat. Even if one could visually confirm that separation of oil from fat is already achieved after centrifugation, the mixing of oil and fat would still take place, likely due to the extremely high affinity of oil to fat cells. Furthermore, many of the commercial systems are messy and cumbersome to use. Since studies showed that contaminating free lipids and RBC could be harmful to the grafted fat, it would be advantageous if one could use a device to fractionate the various components of the fat preparations in a clean and easy way.
The present invention is advantageous in that it provides a system for liquid component fractionation and application method thereof, wherein by operation of a stopcock valve, it is easy and effective to fractionate components of a liquid solution or liquid mixture.
Another advantage of the present invention is that it provides a system for liquid component fractionation and application method thereof, wherein the system functions as a centrifugation tube that allows liquid state components being fractionated, with subsequent sequestration of desired one or more fractions being collected from a collection port, while various components in the liquid have different relative weights.
Another advantage of the present invention is that it provides a system for liquid component fractionation and application method thereof, wherein the system is suitable for the isolation and purification of PRP from whole blood, so that the purified PRP could be subsequently used for various clinical and experimental purposes.
Another advantage of the present invention is that it provides a system for liquid component fractionation and application method thereof, wherein the system for PRP isolation from the blood is easy to use, saves time, increases yields, provides the capability of concentrating PRP up to 15 to 20 folds or even more, facilitates the differential isolation of LR-PRP and LP-PRP, improves the consistency of the PRP production, and avoids the need for additional special equipment purchase because the system of the present invention could easily fit in most standard tabletop centrifuges.
Another advantage of a system for liquid component fractionation and application method thereof of the present invention is that, the system is suitable for the fractionation of fat components, including, but not limited to, removal of contaminants from fat, refinement of nano fat, and purification of fat stromal vascular fraction (SVF).
Another advantage of a system for liquid component fractionation and application method thereof of the present invention is that, due to the fact that the present invention is very versatile in nature, the system could also be used in the preparation of other materials or substances in other fields and industries, such as lipids fractionation, so long as there are differences in relative weights among the fractions in the liquid solution or liquid mixture.
Additional advantages and features of the invention will become apparent from the descriptions that follow, and may be realized by means of the instrumentation and the various combinations of modifications particularly pointed out in the appended claims.
According to the present invention, the foregoing and other objects and advantages are attained by a system for liquid component fractionation, comprising:
a first container having a first containing cavity;
a second container having a second containing cavity; and
a tunnel connecting member, provided between the first container and the second container, comprising at least one tunnel body having one or more connecting tunnels communicating with the first container and the second container, and a collection outlet member, having a collection port, coupled to the at least one tunnel body, and
a stopcock valve, provided at the middle of the tunnel connecting member, having three ports configured for selectively aligning with the one or more connecting tunnels and the collection port.
According to another aspect of the present invention, the present invention further provides a method for liquid component fractionation through a system which comprises a first container, a second container, a tunnel connecting member provided between the first container and the second container, and a stopcock valve provided at the middle of the tunnel connecting member, wherein the method comprises the following steps.
(a) Centrifuge the system which is filled with a liquid to separate the liquid into a plurality of fractionated layers.
(b) Operate the stopcock valve to allow at least one of the plurality of fractionated layers to be collected through a collection outlet member of the tunnel connecting member.
Still further objects and advantages will become apparent from a consideration of the ensuing descriptions and drawings.
These and other objectives, features, and advantages of the present invention will become apparent from the following detailed descriptions, the accompanying drawings, and the appended claims.
The following description is disclosed to enable any person skilled in the art to make and use the present invention. Preferred embodiments are provided in the following description only as examples and modifications will be apparent to those skilled in the art. The general principles defined in the following description would be applied to other embodiments, alternatives, modifications, equivalents, and applications without departing from the spirit and scope of the present invention.
Referring to
The first container 10 and the second container 20 can be embodied as two transparent conical tubes coupled to an “extra-large” 3-way stopcock valve 40. Mechanically and methodologically, the system can function as a centrifugation tube that allows liquid state components to be fractionated, with subsequent sequestration of one or more fractions from a collection port, while the various components in a liquid have different relative weights.
According to this preferred embodiment, the first container 10 comprises a first container body 11 with a first containing cavity 111, and a first cap 12 which can be mounted to the distal end of the first container body 11. The second container 20 comprises a second container body 21 with a second containing cavity 211, a movable member 22 (similar to the plunger tip of a conventional syringe, made of rubber) disposed in the second containing cavity 211, a plug member 23 (similar to the plunger shaft of a conventional syringe) which can be screwed onto the bottom supporting member 222 of the movable member 22 and when operating, can push the movable member 22 in the lower containing body 21, and a cap 24 mountable to the distal end of the second container body 21.
Referring to
The stopcock valve 40 is provided at the tunnel connecting member 30 and comprises a valve plug 41, with preferably a first interconnecting port 401, a second interconnecting port 402 and a third interconnecting port 403 which are selectively aligned with the first containing cavity 111, the second containing cavity 211, and the collecting outlet member 34 respectively, when used for platelet purification. The stopcock valve 40, according to this preferred embodiment, is a valve system that comprises three valve port identifiers 42, a valve lever 43, and an “off” position identifier 44. Where the “off” position identifier 44 points that particular corresponding port will be blocked off for any flow. The three valve port identifiers 42 indicates the respective positions of the three inter-connecting ports 401, 402 and 403 of the valve plug 41.
According to an application of this preferred embodiment of the present invention, the system can be employed for liquid component fractionation, such as PRP isolation from the blood. The system is easy to use, saves time, delivers better yields, provides the capability of concentrating PRP up to 15 to 20 folds or even more, facilitates the differential isolation of LR-PRP and LP-PRP, improves the consistency of the PRP production, and avoids the need for additional special equipment purchase because the system of the present invention could easily fit in most standard tabletop centrifuges.
For PRP extraction, freshly drawn blood is mixed with an anticoagulant, for example ACD (acid-citrate-dextrose) buffer at room temperature. After the first cap 12, which is secured on the first container body 11 (screwed on to the distal end portion of the first container body 11), is unsecured and removed from the first container body 11, the blood is instilled into the system, passing the first container cavity 111, through the connecting tunnel 32 in the middle of the system, reaching the second container cavity 211. The connecting tunnel 32 has an inner diameter, preferably around 6 to 7 mm, smaller than the diameter of the first container cavity 111 and the diameter of the second container cavity 211. Practically, the inner diameter could vary from 3 to 15 mm, depending on the sizes of the first and second containers 10 and 20. In application, the first container 10 can be embodied as the upper container while the second container 20 can be embodied as the lower container, and the blood is filled into the system through the upper container.
The tunnel connecting member 30 can be fortified with a thicker plastic wall (actual thickness depends on the material), for better strength. The thickened wall is provided with a side wing 33 extended from one side of the tunnel connecting member 30 and configured for the purpose of abutting the wall of the centrifugation container for better stability of the system while being centrifuged, as shown in
The connecting tunnel 32 of the tunnel body 31, having a straight elongated configuration, allows the blood to flow downward from the upper first container 10 to the lower second container 20, while the valve lever 43 of the stopcock valve 40 is switched horizontally to block the collection outlet member 34. This collection outlet member 34 of the stopcock valve 40 is provided at the opposite side of connecting tunnel 31 with respect to the side wing 33. The blood then enters the second container 20 through the connecting tunnel 32 and reaching the bottom of the second container 20, which comprises the movable member 22. The movable member 22 comprises a movable member 221 similar to a regular syringe plunger rubber tip top and an engaging member 222 provided at the free end portion thereof for supporting the movable plunger tip 221. Throughout the centrifugation process, the second cap 24 is kept on to securely cover the free end portion of the second container 20, holding the engaging member 222 and providing a definitive mechanism to contain the blood content.
The cap 24 is removed after centrifugation, revealing the engaging member 222 which has inner threads, allowing a separate, detachable plug member 23 to be engaged thereto by the action of twisting-on using its outer threads at the free end portion. Once the plug member 23 is twisted on to the bottom of the member 222, they form the whole plunger complex and could now function fully, allowing precise gliding of the movable member 22 up or down the second container 20 smoothly.
The tunnel connecting member 30 further comprises a sealing cap 35 comprising a central plug 351 which fits into and provides sterility protection to the collection outlet member 34, wherein this connection can be accomplished by the Luer-lock mechanism. The position and the height of this sealing cap 35 are in such a manner that the system abuts the centrifugation container on the opposite side from the side wing 33, for a sturdy and undisturbed centrifugation. As a result, this system is very stable throughout the centrifugation process. Neither the first or the second cap 12 and 24 are air-tight, to enable the movement of liquid contents in either directions by manipulating the stopcock valve 40 only, without the need to uncap the tubes. Additionally, first and second conical slope surfaces 13 and 25 are at the proximal end portions of the first and second container 10, 20 respectively, transitioning from the first container 10 and the second container 20 to the tunnel connecting member 30 respectively, each forming a wide angle with the straight container walls at preferably 120 to 150 degrees (the workable range could be from 100 to 170 degrees), in order to facilitate the movement of the blood components in either upward or downward direction. Accordingly, no unwanted retention of the blood components will take place in the transition sections, minimizing RBC contamination at the end.
Once the blood is filled into the system of the present invention, the system is placed in a standard centrifuge container for centrifugation at 900 g for 8 minutes, at room temperature. And this combination of numbers could vary widely, as have been practiced by many. For example, the g force could be from 300 g to 1500 g, and the time could be 3 to 20 minutes. There should be no braking at the end of the centrifugation, so as not to disturb the “buffy coat”.
Thereafter, referring to
To isolate the platelets, the “buffy coat”, which contains platelets and leukocytes, needs to be physically separated from RBC. This is achieved by slowly pushing the plug member 23 of the plunger complex further up, pushing the “buffy coat” to pass the boarder of the first port 402 which is the bottom port of the stopcock valve 40.
Next, further referring to
Since LR-PRP and LP-PRP were found to have distinctively different indications in the treatment of musculoskeletal diseases, one could easily harvest LR-PRP or LP-PRP by manipulating the position of the “buffy coat” at the threshold of the lower valve port of the stopcock valve 40, as shown in
Both
To concentrate the platelets in the PRP to a higher level, the PPP could be suctioned out with minimal disturbance from the top of the first container 10 with the top first cap 12 removed. Because the stopcock valve 40 effectively closes the contaminating RBC off at the bottom, as shown in
This middle section of the system of the invention comprises the stopcock valve 40 and the tunnel connecting member 30. The rotatable stopcock valve 40 is housed in the tunnel connecting member 30. The stopcock valve 40 has three interconnecting ports 401, 402 and 403. The tunnel connecting member 30 has a first passage 321 and a second passage 322, and a collection port 341. At any point of time, only two of the valve plug ports 401, 402,403 are open to each other. The stopcock valve 40 further comprises a blocking member 45 (
The valve plug 41 is essentially a cylindrical structure hugged snugly by the outside valve body. Of course, the plug handle is not encased by the tunnel connecting member 30. The tunnel connecting member 30 further comprises an annular rib 36 on the inner surface of the tunnel connecting member 30, which presses against the annular wall structure on the valve plug 41 into a sealing engagement with the wall of the tunnel connecting member 30 forming a watertight seal, as shown in
On the end portion of the tunnel connecting member 30, opposite to the collection outlet member 34, is a disk shaped and slightly raised round prominence 37 and on the end of the valve plug 41 is a slightly larger and round recession area 411, as shown in
Compared to various commercially available kits or methods, this invention is inexpensive, easy to use, smooth in operation, and safe to handle. It does not require a long time to obtain the final product(s), has a high yield with consistent results, and shows minimal disturbance during the harvesting stage. Because the system of the invention fits in most readily available table-top centrifuges, there is no need to buy additional expensive special centrifuge. Additionally, the contamination by RBC is minimal, and the ability to concentrate PRP up to 20 times in one spin is a powerful feature. Last but not the least, it is a significant advantage that the device could be utilized to differentially isolate LR-PRP and LP-PRP.
Depending on the requirements, the volume handled could be anywhere from 5 ml to 100+ ml. Of course, volumes at the more extreme ends need to be fitted into specially designed tubes.
The g force required for centrifugation is mostly a personal preference, so long as the numbers are not too much off. Note that one of the more important features to pay attention to is the angle in
The basic structure, such as the first and second container which are embodied as tubes in this preferred embodiment, could also be further modified. As long as the basic principles are observed, they could be made detachable to further facilitate the separation of samples requiring special treatments.
In summary, the present invention is simple to use and inexpensive to manufacture. The system of the present invention is adapted to be a disposable device after a single use, to avoid cross contamination. Sterilization post manufacturing is achieved by gamma-radiation or similar measures. Despite the mentioning of only PRP in the above text, it is by no means limited to these uses. In addition to PRP isolation, various fat preparations could be fractionated. In fact, anything that present differential relative weight qualities in a sample material could be separately isolated using this invention.
As an Example of Blood fractionation for PRP preparation, a phlebotomist may draw blood into a syringe containing 1/10 volume of ACD or CPD buffer. The volume of blood needed depends on the usage. For most treatments, 20 ml of blood is usually sufficient. The blood is gently filled into the system from the top, with the valve lever 43 of the stopcock valve 40 blocking the collection port 341. Once the blood is filled into the tube complex of the system, the system is placed into a standard centrifuge container for centrifugation at 900 g for 8 minutes at room temperature without braking. Then, the system is removed from the centrifuge after it comes to a complete stop.
Then, at this point, the second cap 24 is removed and the plug member 23, comprising a plunger shaft, is fastened to the engaging member 222 to assemble the complete plunger complex which comprises the movable plunger tip 221, the engaging member 222 and the plug member 23. The plug member 23 is then pushed gently to let the rubber head glide upward smoothly. Consequently, the “buffy coat” migrates toward the tunnel connecting member 30, reaching the stopcock valve 40, as shown in
To isolate the platelets, the “buffy coat” which contains platelets and leukocytes which need to be physically separated from RBC. This is achieved by slowly pushing the plug member 23 further up, pushing the “buffy coat” to pass the boarder of the lower valve port of the stopcock valve, as shown in
Next, the collection syringe 50 is connected to the collection outlet member 34. The valve port of the stopcock valve 40 is then turned to block the RBC compartment in the second containing cavity 211. This is a very easy-to-perform motion, and no disturbance to the “buffy coat” should take place. After the valve is shut to the RBC port, the PRP could be easily collected by withdrawing the plunger of the collection syringe 50. Note that no RBC from the lower tube compartment could enter the collection port or the syringe once the three-way stopcock valve 40 locks down the passageway to RBC.
Additionally, it is easy to harvest LR-PRP or LP-PRP by manipulating the positions of the “buffy coat” at the threshold of the lower valve port of the three-way stopcock valve 40, as described earlier.
As another example of the application of the system of the present invention,
The fractionation starts with a centrifugation with the same setting as in the above second example and
Traditionally, it has always been a difficult task to harvest SVF in a clean way. While the numbers of SVF cells are in the ranks of 105 to 106 per ml of fat, the single cell numbers in the pale-colored layer and the fine nano fat layers are in the ranks of 109. Even a diminutive, almost ignorable amount of contamination of the SVF by the above-mentioned fractions would drastically undermine the validity of the number of the SVF cells counted at the end, and therefore, the subsequent treatment regimens or related experiments. This kind of contamination takes place very easily and very frequently with most, if not all, of the mechanical systems currently in use, for reasons such as the contaminating fractions sticking to the walls of the tube/bottle, or some almost invisible small pieces breaking away from the main fraction layer during the SVF collection. These are further complicated by the fact that the morphology of pre-adipocytes and small fat cells are not much different from the SVF cells when in suspension. Considering these, it is no wonder that the numbers of harvested “stem cell” from fat vary from 104 to 2.24×107 among different publications. And this fact significantly hampers the progress in the field of fat stem cell research and clinical application. As far as we know, currently, there is no method or commercially available mechanical system for the purpose of purifying SVF without any risk of cell contamination by the other fractions.
With the present invention, by manipulating the valve positioning, it is easy to flush out the pelleted SVF. The only requirement is to pre-calculate the amount of water that might exist in the final preparation, so as to let the water occupy the whole second container 20 and some on the lower portion of the first container 10.
Theoretically, there are much more SVF cells in the fat than the small number of cells existing in the pellet, as shown in
In any case, since the SVF need to be flushed out and re-pelleted for harvesting, it might be more convenient to use a first alternative mode of the above preferred embodiment, as shown in
Phase-contrast microscopic photos in
More specifically, as shown in
As shown in
As shown in
Specifically, the port 401E facing the second container 20, when the valve lever 43E is aligned with the collection outlet member 34, could be modified.
One skilled in the art will understand that the embodiment of the present invention as shown in the drawings and described above is exemplary only and not intended to be limiting.
It will thus be seen that the objects of the present invention have been fully and effectively accomplished. The embodiments have been shown and described for the purposes of illustrating the functional and structural principles of the present invention and is subject to change without departure from such principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the following claims.
