WO2015174871A1 - Isolation of adipose derived cells - Google Patents

Abstract

There is described xeno-free methods, devices and systems for isolating cells from adipose tissue, the method comprising: a) providing a sample of adipose tissue; b) optionally, evaluating the sample to ensure it complies with pre-determined criteria; c) optionally, mincing the sample into smaller pieces; d) washing the sample with a decontamination agent; e) dissociating the matrix of the adipose tissue sample using a matrix dissociating agent; f) isolating the nucleated cells; g) purifying the nucleated cells; and h) optionally, evaluating the cells of the sample to ensure they comply with pre-determined criteria, wherein the method is carried out such that no non-human biological components are introduced into the sample, and wherein isolated cells are produced. Also described are isolated cells produced by the above method.

Description

Isolation of Adipose Derived Cells

Field of the Invention

The present invention relates to xeno-free methods, devices and systems for isolating enriched regenerative clinical grade cells from adipose tissue that are suitable for human therapeutic use. Further methods comprise xeno-free cryopreservation of the isolated cells.

Background to theinvention

Adipose tissue, as an organ, has been attracting the attention of the medical and scientific community for its vast regulatory functions. Its proteins are secreted to act in functions of different body tissues, not only over its metabolism, but also in inflammatory and immunity mechanisms. Attention has been brought to the importance of these on promoting regenerative and tissue tolerance, characteristics of major importance on efficiency of cell therapy, tissue engineering and regenerative medicine. At the cellular level, the adipose tissue is composed of adipocytes and a heterogeneous fraction of cells, named Stromal Vascular Fraction (SVF). Several subpopulations have been identified, including adipocyte progenitors, endothelial progenitors, macrophages, lymphocytes, monocytes, fibroblasts, pericytes and also hematopoietic and mesenchymal stem cells, with the latter - adipose derived stromal/stem cells - the most studied for their potential to differentiate into mesoderm lineages (bone, cartilage and fat, among others). Given the multivariate functions of adipose tissue, stromal vascular fraction represents a minimally manipulated cell niche with processing advantages and likely higher therapeutic potential, contributing to a better treatment response - meeting the specific physiological conditions - as compared with the use of the stromal/stem cell population (Alexander RW, J Prolotherapy. 2012, 4:855-69; Bourin P. et al, Cytotherapy, 2013, 15:641-648).

The therapeutic use of SVF, as an alternative to its mesenchymal stem cells, has some practical advantages: i) safety, by avoiding extensive manipulation of the cells, the risk of contamination is considerably reduced; ii) speed - the high cell yield allows a faster therapeutic solution, avoiding consuming of further time or resources in cellular expansion. This therapeutic rational has raised high clinical interest, leading to its implementation in clinical trials including therapies for lipodystrofia (NCT00715546), myocardial infarction (NCT00426868, NCT00442806, NCT01216995), diabetes type I and II (NCT00703599, NCT00703612), recto-vaginal and enterocutaneous fistulas (NCTO 1548092, NCTO 1584713) and liver cirrhosis (NCT00913289, NCTO 1062750). In addition to these clinical studies, SVF has been proposed for chronic diseases, such as Crohn's disease, graft versus host disease, autoimmune diseases such as multiple sclerosis, allergic conditions and immunological inflammatory diseases such as arthritis.

The worldwide growing industrial interest of adipose derived cells for therapeutic applications is also reflected by the increase in patent filings relating to systems, methods and devices, both for manual and automatic processing of adipose tissue for isolation of its cells (US2013/0122584 Al, US2013/0034524 Al ; US2012/0264200 Al; WO2013/030761 Al). The isolation protocol has a great impact on the ultimate composition, yield and quality of the cells obtained.

One factor contributing to the variability of the cells reported in the literature arises from the reagents used, as many of these comprise components of animal origin, which severely limits therapeutic application of the final product. There remains an outstanding clinical demand for methods using systems and reagents approved for human use which meet the criteria of good manufacturing practice (GMP) and free of any non-human biological component (xeno-free). The use of human xeno-free components ensures much lower risk of animal pathogen contamination, no interference of animal (non-human) sera on cell characteristics and behaviour, and, not less important, contribute to decreased ethical issues regarding the use of animal derived components.

Isolation procedures include generally the following sequential steps: 1) harvesting of the adipose tissue; 2) transportation of adipose tissue from harvesting site to processing facilities (with exception to automated systems which are used at harvesting site); 3) pre-processing quality control assessment; 4) washing of the adipose tissue; 5) dissociation of tissue matrix to release cells (commonly performed by digestion of the tissue at 37°C); 6) differential centrifugation; 7) post-processing quality control assessment; and 8) cell application.

Document CN102433299 A discloses a method for separating, culturing and purifying mouse adipose-derived stem cells. This method is limited to the processing of cells from four week old male mice. Furthermore, the digestive step involves the use of fetal bovine serum as a component of the digestive solution. In the context of the present invention, this would obviously introduce the risk of animal pathogen contamination as well as ethical issues regarding the use of animal-derived components. Accordingly, this document does not disclose a xeno-free process for isolation of clinical grade cells from adipose tissue suitable for therapeutic use in humans.

Document Carvalho, P.P. et al, Tissue Engineering: Part C, 2013, 19(6):473-78 discloses the use of xeno-free enzymatic products for use in the isolation of human adipose-derived stromal/stem cells (ASC). While culture of ASC may be carried out in a culture medium containing fetal bovine serum, it is mentioned that previous work showed comparable results under serum-free and xeno-free conditions (Lindroos, B. et al., Cytotherapy, 2009, 1 1(7): 958-72). However, after the enzymatic digestion of lipoaspirate and centrifugation, the SVF is re-suspended in a medium containing 10 % fetal bovine serum. Accordingly, this document does not describe a genuinely xeno- free process as disclosed in the current invention.

Document WO2009/1 16088 A2 describes the use of a composition for use in patients undergoing organ transplant, the composition consisting of a combination of adipose tissue-derived mesenchymal stem cells, bone marrow-derived haematopoietic stem cells and peripheral blood stem cells. The document describes an essentially xenogenic material-free technique for collecting and culturing mesenchymal stem cells from adipose tissue. The process as described is carried out at 37 °C, which is in fact not optimal. According to the present invention, after tissue digestion, the cell suspension is added to a xeno-free protein solution and processed at a much lower temperature of 4 °C to further inactivate any remaining enzyme activity. This temperature is maintained during the remaining process until cryopreservation, demonstrated to be advantageous towards preventing cell aggregation and leading to a much higher product yield.

Document WO2006/014156 Al describes systems and methods for isolating and using clinically safe adipose-derived stem and/or progenitor cells. The document reveals a device for separating and concentrating cells from a variety of tissues, including adipose tissue. However, the document does not mention the advantageous xeno-free characteristics of the present invention or reveal that the disclosed system is adequate for isolation of the preferred cell sub-populations herein disclosed. Furthermore, on page 29, lines 1 1-22, it is revealed that the regenerative cell composition may also contain one or more contaminants carried over from the adipose tissue fragments. Accordingly, the process of the present invention is clearly superior in terms of assuring greater product purity, as can be observed from the picture in the lower right quadrant of Figure 4.

Current adipose derived cell isolation systems require immediate therapeutic application of the cellular product, due to the lack of validated preservation and storage procedures, either xeno or xeno-free, for this heterogeneous population. Current art relates to cryopreservation systems for the adherent fraction of cells, excluding the non-adherent (WO2013/040649 Al, WO2013/020492 Al). The need for immediate use of the adipose derived cells causes increased burden to the patient and physician, since a double step intervention is always needed at the moment of care: firstly, an initial surgical procedure for adipose tissue harvest, including a waiting period for cell isolation (either through manual procedure or automated equipment); followed by a second intervention for therapeutic cell administration. This approach raises questions regarding the implementation of quality control methods, as the time available for its application under a clinical setting is usually rather limited and may limit its efficacy in rejecting non-compliant samples and/or products.

An improved method would involve tissue harvesting and subsequent processing for therapeutic delivery. As harvesting and therapeutic delivery occur in different moments in time, more stringent and effective quality control methods can be implemented, increasing safety for the patient. Furthermore, by delivering cells cryopreserved, therapeutic planning can be performed in advance by the physician and patient, once cells have been previously characterized and/or selected (targeted and personalized therapy), and the therapeutic action can be considerably simplified, controlled and expedited, improving efficacy while reducing costs, time and burden. The significance of a xeno-free cryopreservation and cell recovery procedure which ensures high cell viability and initial cell population characteristics is invaluable.

SVF can be used as a minimally manipulated, ready-to-use platform for further cell operations, including expansion, differentiation, among others. The availability of cryopreserved SVF would enable subsequent manipulation steps not to occur immediately after SVF isolation, but after a significant amount of time such as days, weeks, months, or even years. This possibility is of great interest as it enables a cryopreserved cell source for repeated patient treatment, as it minimises variable costing related with: tissue harvesting - avoids repeated surgery; and quality control methods related with qualification of starting material - a single starting material is qualified. In addition, SVF can be used directly or can be used as a starting material for several cell based products. In addition to cell characterization for potency, quality control for evaluation of safety aspects is equally critical. Microbiological contamination of the collected adipose tissue is far more common than desired, mostly by commensal skin bacteria, due to high contact with the donor's skin during the collection procedure. By delivering cells in a cryopreserved state, appropriate quality control can be performed beforehand, to ensure safety of the therapeutic product, which should be free of bacterial contamination, mycoplasma, viral agents and pyrogen-free. Regarding the cell isolation process, there is a need to improve towards procedures that eliminate such microbial load, while preserving quantity, viability and integrity of the tissue. Contamination of the cellular product with undesired or unsafe components such as tissue debris, oil droplets, cell agglomerates, red blood cells or other residues from solutions used during cell isolation might also be unsafe or inadequate for clinical application. Furthermore, the presence of such contaminants within the cellular product interferes with cell characterization which is carried out by quantification of parameters such as cell number, cell viability and cell immunophenotype. Contaminants can ultimately cause misleading outcomes (WO2014/015229 Al). Several approaches are used for cell isolation, yet doubts still remain regarding the impact on the final cell composition (Bourin P. et al , Cytotherapy, 2013, 15:641-648). Novel cell isolation systems and methods should include purification steps that result in a highly purified and enriched cellular product, maintaining initial cell characteristics.

Furthermore, variable nucleated cell yield and/or cell viability is obtained from different systems, which compromise therapeutic efficacy, either by insufficient cell dosage or inappropriate cell condition status. To avoid this, and to provide better solutions for patients, it is necessary to use a complete and integrated system composed of integrated processes for tissue collection and transportation, cell isolation, cryopreservation and recovery, as well as reliable cell characterization methods, resulting in consistent cell yield and reproducibility among tissue sources and high viability of the therapeutic cellular product.

Current adipose-derived cell isolation systems present limitations on scalability of adipose tissue processing, as well as lack of flexibility in manipulating distinct adipose tissue sources, which result in limited sourcing of patients' therapeutic cells. This can compromise or reduce efficacy, as well as efficiency, of the clinical approach. An improved system for cell isolation and cryopreservation should be scalable, while simultaneously maintaining process efficacy and end-product characteristics. Furthermore, some systems use complex and/or expensive components or materials which are reusable between processing of two independent adipose tissue samples, ultimately increasing risk of cross-contamination. The use of disposable and cost competitive materials and devices throughout the complete system is also desirable.

For compositions of clinical grade cell fraction, the characteristics of the cells' subpopulations are critical for therapeutic efficacy. Current knowledge values cells which are positive for the expression of CD34+ surface marker for their therapeutic potential, yet its relative amount within the isolated cell population varies, depending on tissue collection and processing (Bourin P. et al, Cytotherapy, 2013, 15:6 1-648). A system that yields a cellular product enriched with these CD34+ cells, in a reproducible and consistent manner, is of great value.

In light of the above challenges, a system which addresses some or all of the problems would be advantageous. In particular, a system which synergistically addresses the limitations and improves the outcomes of the enriched regenerative clinical grade cells would be of significant value for cell therapy development and application.

Summary of the Invention

The present invention relates to methods, devices and systems for isolating and cryopreserving an enriched population of regenerative clinical grade cells from adipose tissue compatible for human therapeutic use, although the same principles can be applied to other mammalian animals.

In a first aspect, the present invention provides a method of isolating cells from adipose tissue. These cells can be clinical grade cells. The method of isolating cells from adipose tissue comprises:

a) providing a sample of adipose tissue;

b) optionally, evaluating the sample to ensure it complies with pre-determined criteria;

c) optionally, mincing the sample into smaller pieces;

d) washing the sample with a decontamination agent;

e) dissociating the matrix of the cells of the sample using a matrix dissociating agent;

f) isolating the cells;

g) purifying the cells; and

h) optionally, evaluating the cells of the sample to ensure they comply with predetermined criteria,

wherein the method is carried out such that no non-human biological components are introduced into the sample, and wherein isolated cells are produced. In the method, no non-human biological components are introduced into the sample. This means that the decontamination agent used to wash the sample and the matrix dissociating agent do not contain any non-human biological components. This results in high quality clinical grade cells which are not contaminated by non-human biological material. The presence of non-human biological material can cause complications when cells are introduced into a patient, for example, by triggering an undesired immune response, or by causing pathogen infection.

Compositions and solutions which are free from non-human biological components are designated as 'xeno-free'. For example, a xeno-free protein solution may comprise human serum, human albumin, and/or recombinant human albumin but it will not contain any non-human biological components such as bovine serum albumin.

'Non-human biological components' which should not be introduced into the sample include immunogenic substances such as, but not limited to, non-human proteins, bacteria, viruses, fungi, spores and endotoxins.

Put another way, the only substances which should come into contact with the sample are substances which are safe for human contact and which will not cause unwanted side effects if the sample was to be introduced into a human patient. Such side effects include an immune response and infection. The safe substances should be non- immunogenic. Safe samples include, but are not limited to, protein solutions which comprise human serum, human albumin, and/or recombinant human albumin.

A number of steps in the method are described above as being optional. In preferred embodiments of the invention, one or . more of the optional steps will be included so that they are not optional.

The method comprises the steps described above. However, this does not mean that the steps have to be carried out in the order specified. The method can also be carried out with some of the steps in a different order and such methods are covered by the method of the invention. For example, the evaluation of step b) does not have to be carried out directly on the sample before the mincing step c). Further, the decontaminating washing step d) can be carried out before, at the same time as, or after the matrix dissociating step e).

The method may further comprise optionally cryopreserving the cells. This may involve:

i) suspending the cells within a xeno-free cryopreservation solution;

ii) cooling the suspended cells in a controlled rate freezer; and

iii) storing the frozen cells within a vapour phase liquid nitrogen tank.

Cryopreservation is important for ensuring the isolated cells remain stored in high quality so that they are of a clinical grade for use with patients. Xeno-free cryopreservation of the isolated cells allows use of the same batch of cells in multiple therapeutic administrations, along a therapeutic timeframe, if required. Furthermore, cryopreservation of isolated cells allows maintenance of their native characteristics during an appropriate period of time. During this period, which might take several days or weeks, validation of cell features is made according to safety and quality criteria, before respective release is made before final therapeutic use.

The method comprises optionally evaluating the sample to ensure it complies with pre-determined criteria. The evaluation may relate to transportation unit temperature, time period from tissue harvesting to processing, donor eligibility, physical integrity of adipose tissue, matching of barcode labelling identification between adipose tissue sample and donor documentation.

The method comprises washing the sample with a decontamination agent. This results in removal of undesired lipids, peripheral blood components and surgical fluids. It also results in the sample being sterilised. Preferably, the tissue is washed with a decontamination agent multiple times. Preferably, the tissue is washed 3 to 8 times. The tissue may be washed 5 times.

The decontamination agent may be an anti-microbial agent which kills any microorganisms in the sample. However, the decontamination agent should not damage the adipose tissue cells in any way. The decontamination agent may be a solution comprising one or more agents selected from vancomycin, gentamicin, cefotaxime, and amphotericin B deoxycholate. The vancomycin, gentamicin and cefotaxime may be in concentrations between 50 and 300 mg_ss/L and amphotericin B deoxycholate may be in concentrations between 10000 and 30000 UI/L as antibiotic/antifungal agents; preferably between 115-140 mg_ss/L and 13500-16500 UI/L, respectively.

The sample can be washed using any suitable amount of decontamination agent. The decontamination agent may be used at a ratio of 1 :2 to 2:1 vol/vol relative to the sample. Preferably it is used at a ratio of 1 : 1.

The method comprises dissociating the matrix of the cells of the sample using a matrix dissociating agent. This can be achieved by using a dissociation solution containing active agents such as enzymes. Suitable enzymes include, but are not limited to, collagenase class I and II, and neutral proteases. Concentrations ranging between 0.1 and 0.8 PZ-U/mL, preferably between 0.3 and 0.4 PZ-U/mL, may be used.

The matrix dissociating agent may further comprise basal cell culture media. In addition, the matrix dissociating agent may further comprise a decontamination agent.

The amount of matrix dissociating agent used may be at a ratio of 1 :3 to 3:1 vol/vol relative to the sample. Preferably it is used at a ratio of 1 : 1.

To cause dissociation of the matrix, the sample is preferably incubated with the dissociating agent. It may be incubated at 10 to 38°C. Preferably, it is incubated at 20 to 38°C. More preferably, it is incubated at 30 to 38°C. More preferably still, it is incubated at 35 to 38°C. Most preferably, it is incubated at about 37°C.

Incubation may take place for a suitable time to cause dissociation of the matrix. This may be between 5 minutes and 180 minutes. Preferably, it is for between 10 minutes and 150 minutes. More preferably, it is for between 15 minutes and 120 minutes. Most preferably, it is for between 45 minutes and 90 minutes. Preferably, the sample is agitated during the matrix dissociation step. Agitation can be between 10 and 300 rpm. Preferably, agitation is between 20 and 200 rpm. More preferably, agitation is between 100 and 150 rpm.

The dissociating step may also comprise straining the sample through a mesh to separate any intact fragments.

The sample may then be centrifuged to separate the cells from any other remaining components.

The method comprises isolating the cells. This can be done by the staged removal of dissociated tissue components following centrifugation. The cell pellet may be recovered and resuspended in xeno-free protein solution. Preferably, the xeno-free protein solution is cold, e.g. 2-10°C. Xeno-free proteins include, but are not limited to, human serum, human albumin, or recombinant human albumin. These may be at a concentration of between 0.1 to 40%, preferably between 1 % and 20% w/v.

The cell suspension may then be refrigerated to 2 to 10°C, preferably to 4 to 8°C. The cells are preferably maintained at 2-10°C for all subsequent steps in the method until cryopreservation, if it is carried out.

The use of cold xeno-free protein solution and/or refrigeration helps to ensure maintenance of the cells' native characteristics, given that, at low temperatures, cell metabolism and cell-cell interaction is diminished.

The method comprises purifying the cells. This is done to remove contaminants or unwanted components from the sample. For example, the cell suspension may be sequentially filtered using different mesh size filters, e.g. using strainers with mesh sizes below 130μηι, 90μηι and/or 50μπι.

Further purification may involve removing red blood cells from the sample. This can be done by using a red blood cell (RBC) lysis buffer and centrifuging the sample to remove haemoglobin released by the red blood cells. The sample can be contacted with the red blood cell (RBC) lysis buffer for 1 to 10 minutes, preferably 3 to 5, and further washed. The washing buffer may also comprise xeno-free proteins.

The method comprises optionally evaluating the cells of the sample to ensure they comply with pre-determined criteria and/or to ensure that the isolated cells are of sufficient quality. Quality control may include the determination of, but not limited to, microbial, endotoxin and mycoplasma contamination. Further, it may include determination of, but not limited to, cell number, cell viability and cell immunophenotype .

In some embodiments, the method produces a sample having a cell number of higher than 150,000 nucleated cells/gram of adipose tissue. The cell viability may be higher than 80%. The cell immunophenotype may be determined for one or more of CD34, CD90, CD105, CD73, CD31, CD45, CD235a, CD44, CD29, CD13, CD10, CD26, CD36, CDl lb, CD49d, CD49e, CD49f, CD106, CD144, CD133, CD146, HLA-ABC and PODXL. The cells produced may have concomitant expression of CD34+CD45- CD31- of higher than 30%. The concomitant expression of CD34+CD90+CD105+ CD73+ may be higher than 15%.

The method may further comprise monitoring the sample throughout the method to ensure compliance with pre-defined acceptance criteria.

The method may further comprise recording information relating to the sample to enable tracing of the sample.

Preferably, the method is carried out under sterile conditions.

Before the step of providing a sample of adipose tissue, the tissue has been harvested from a patient. Preferably, after harvesting the sample is maintained between 0 and 15°C, preferably between 2 and 8°C. Preferably, the method is carried out on the sample within 48 hours of the sample being harvested. In another aspect, the present invention provides isolated cells produced by the method described above. These cells can be used therapeutically and administered to a patient. In a particular embodiment, the method of isolating cells from adipose tissue comprises:

a) providing a sample of adipose tissue;

b) washing the sample with a decontamination agent;

c) dissociating the matrix of the cells of the sample using a matrix dissociating agent;

d) isolating the cells; and

e) purifying the cells;

wherein the method is carried out such that no non-human biological components are introduced into the sample, and wherein isolated cells are produced.

The method comprises a tissue collection device, and transportation unit, and a quality management system. The method continues at the clean-room, GMP compliant tissue processing facilities, where liposuction surgical fluids are removed, or lipectomy tissue is mechanically minced into smaller pieces, both further washed with decontamination solution. Adipose tissue is further dissociated with matrix dissociation solution to release the nucleated cells. Dissociated tissue is further filtered and centrifuged to obtain the nucleated cells. To recover the cells from remaining components of dissociated tissue, a staged removal of the layers of oil, dissociation solution and cells is performed to avoid contamination of cells with oil, further leading to interference on cell characterization. Cells are then recovered into a cold xeno-free protein solution, and purified involving steps of filtration, centrifugation and lysis of residual red blood cells. The purified clinical grade cells are sampled for quality control, including but not limited to, cell counting, viability assessment, immunophenotypic analysis and cell culture. Finally, cells are concentrated by centrifugation, where supernatant solution is collected for sterility testing, and the cell pellet is stored in refrigerated conditions for further applications, such as, but not limited to, cell culture, immediate therapeutic application or cryopreservation. In another embodiment the invention relates to a method for cryopreservation of clinical grade cells from adipose tissue. This method comprises a cryopreservation storage device, a controlled rate freezer, a cryopreservation storage system and a quality management system. After obtaining adipose tissue cells, the cryopreservation method continues at the clean-room, GMP compliant tissue processing facilities. A cold cryopreservation solution is prepared, immediately before use, composed by a xeno-free protein solution and a cryoprotectant. Cells are dispersed within cryopreservation solution at pre-defined concentration, stored within the cryopreservation storage device and further cryopreserved by the use a preprogrammed controlled rate freezer. Cryopreserved cells within storage device are further stored within a vapour phase liquid nitrogen tank. The cryopreservation storage system further requires maintenance of cells under quarantine location until release after acceptance of all quality control parameters.

In a preferred embodiment of the present invention, the adipose tissue is human, of any anatomical location, obtained by lipectomy or liposuction, wherein the tissue is processed to obtain clinical grade nucleated cells for application in research or in therapies.

The foregoing summary of the invention, as well as the preferred mode of use and further advantages thereof, will be best understood with reference to the following detailed description of embodiments, when read in conjunction with reference to the figures.

Brief Description of the Figures

Figure 1 illustrates a system according to the present invention. The reference numbers are as follows:

1. system for isolating and cryopreserving clinical grade cells;

2. quality management system;

3. database;

4. adipose tissue collection system;

5. cell isolation system;

. cell cryopreservation system; 7. cell product release system.

Figure 2 is a flowchart of the method for isolation and cryopreservation of clinical grade cells in accordance with the present invention.

Figure 3 illustrates a preferred embodiment of the adipose tissue transportation unit. The reference numbers are as follows:

10. Transportation unit;

1 1. Tissue collection set;

1 1a. Tissue collection device;

1 lb. Tissue collection adaptor;

12. Cold accumulators;

13. Isothermal box;

14. Separator;

15. Temperature logger;

16. Leak-proof specimen transport bag for tissue collection device;

17. Absorbents;

18. Leak-proof specimen transport bag for blood collection devices;

19. Blood collection set;

20. Absorbent;

21. Barcode labels;

22. Envelope with documentation.

Figure 4. Qualitative evaluation of isolated cells. Top - microscopic observation (at two depths of focus) of cell suspension before purification, containing: oil droplets (a), cell aggregates/tissue debris (b), red blood cells (c) and single nucleated cells (d). Bottom left: Cell pellet before and after red blood cell lysis. Bottom right: microscopic observation of cell suspension after purification, containing single nucleated cells and depleted of contaminants (a), (b) and (c).

Figure 5. Quantification of red blood cells (RBC) present in peripheral blood as well as unpurified and purified cells isolated from adipose tissue. Figure 6. Cell viability, expressed as percentage of live cells, as well as cell yield, expressed as number of cells per gram of adipose tissue, obtained with or without purification steps.

Figure 7. Concomitant expression of surface markers of cells obtained with or without purification steps.

Figure 8. Cell viability, expressed as percentage of live cells, obtained after cryopreservation when using human protein or animal serum as protein component of cryopreservation solution.

Figure 9. Concomitant expression of surface markers of cells cryopreserved with solution containing human or animal proteins.

Figure 10. Cell viability, expressed as percentage of live cells, obtained before and after cryopreservation with human protein.

Figure 1 1. Concomitant expression of surface markers of cells before and after cryopreservation with human protein.

Figure 12. Cell yield, expressed as number of cells per gram of adipose tissue,, obtained from liposuction or lipectomy samples.

Figure 13. Concomitant expression of surface markers of cells obtained from liposuction or lipectomy samples

¾eta¾yij¾ Invention

One skilled in the art would understand the following description, as well as terminology used herein, as to best describe the invention, and embodiments chosen to do so are not intended to be exhaustive or to limit the invention to the form disclosed. It should furthermore be understood that figures are provided for illustration purposes, and should not be considered as a definition of the limits of the present invention. In a first embodiment, the invention relates to a system for isolation and cryopreservation of adipose-derived clinical grade cells. Referring to Figure 1, the system 1 includes a quality management system 2, a database 3, an adipose tissue collection system 4, cell isolation system 5, cell cryopreservation system 6 and a cell product release system 7. The database stores, in a predefined organized manner, all information related to an adipose tissue sample, registered throughout all systems of Figure 1.

In another embodiment, the invention is related to a method for isolation of clinical grade cells from adipose tissue, collected by liposuction or lipectomy procedures (Figure 2). This method starts by ensuring, through the quality management system, that there is a database with an encoded program to organize and store all information related to a specific adipose tissue sample, for traceability in accordance to guidelines and regulations applicable to transplantation units and medicinal products. Each sample is identified by a unique code, which results in the anonymising of donor information, whereas in a preferred embodiment, a barcode labelling system is used for identification. By all information related to a sample, is understood: i) all material and reagents used from harvesting to end application; ii) all documentation related to donor, adipose tissue sample, materials and reagents; iii) all data generated throughout isolation and cryopreservation systems, related to adipose tissue (AT) sample and cellular product. This method continues by ensuring, that: i) all materials and reagents used are appropriately marked and/or GMP-compliant, both with certified sterility, and/or internal sterility control; ii) all materials that contact with biological tissue and/or fluids, and/or cells are disposable; iii) all intermediate devices used for storage of biological tissue and/or fluids, and/or cells are barcode labelled with sample identification code. All components used are tracked for lot, expiry date, manufacturer and supplier.

As used herein, "liposuction" and "lipectomy" are surgical procedures performed to remove deposits of fat located under the skin. Liposuction may be performed by, but not limited to, the following techniques: suction-assisted liposuction (SAL), power- assisted liposuction (PAL), ultrasound-assisted liposuction (UAL), twin-cannula (assisted) liposuction (TCAL or TCL), water-assisted liposuction (WAL), external ultrasound-assisted liposuction (XUAL or EUAL), laser assisted or tumescent technique. Lipectomy consists on the surgical resection of fat, generally including removal of adjacent excess of skin. The cell isolation process (Figure 2) initiates by assembly and supplying (30) of an adipose tissue collection device within a transportation unit to the authorised harvesting facility (example: hospital or clinic). In a preferred embodiment (Figure 3), the transportation unit (10) contains at least, but not limited to: an adipose tissue collection device (11), cold accumulators (-18°C / -20°C) (12), within an isothermal box (13), and separated by a plastic separator (14), a temperature logger (15), leak- proof plastic bags (16), (18), absorbent material (17), (20), blood tubes and blood collection set for donor blood sampling (19), identification (ID) barcode labels, expiry date label and security seal label (21) and documentation (22). In a preferred embodiment, documentation includes: a letter for the medical doctor, an informed consent (a copy for donor and a copy for the processing facility), a medical questionnaire, the adipose tissue collection procedure, the adipose tissue collection report and a donation registry. In a preferred embodiment, the transportation unit is composed of an isothermal cardboard box; an internal separator; an informative outer box, indicating unit and service company information. The transportation unit should be approved by the National Authority Agency, for transportation of biological tissues and/or fluids.

The preferred embodiment for adipose tissue collection device (ATCD) is one of which physicians are familiar with in their daily surgical duties, in order to minimize time at the point of procedure, as well as learning curve of all clinical staff involved, for tissue harvesting, such as single-use canister. The tissue collector should be an appropriate medical device; sterile and approved for storage of biological components for clinical applications. In a preferred embodiment, each ATCD can store up to 1000 mL in volume of collected adipose tissue. The ATDC is then placed within the leak- proof plastic bag with absorbent material to restrain adipose tissue and fluids in case of leakage. After adipose tissue collection (31), transportation unit is sent to GMP facilities (32). At GMP facilities, quality control (QC) is performed (33). The first step relates to inspection of the transportation unit, and registry on a standardized form, including, but not limited to: a) confirmation of the transportation unit expiry date is after the unit's arrival date; b) confirmation of the presence and integrity of security seal; c) integrity of the shipping container; d) confirmation of collection device with AT within security bag with absorbent - annotation of volume collected and signs of leakage; e) confirmation of blood tubes within security bag with absorbent; f) confirmation of barcode label on the collection device with adipose tissue and on the blood tubes; g) the temperature logger has registered temperature values within the range 2°C-8°C, between cooling period and reception; h) confirmation that the AT was collected within the past 48h; i) confirmation of documentation and complete registry of information and g) confirmation of packaging performed as instructed. If conditions of b) or c) fail, or in condition d) there are signs of leakage AT sample is discarded as biohazard waste and registered as so at database, for tracking purposes. If any of the other conditions fail, sample will be held in quarantine until risk assessment is made for the compliance of final product. If conditions are met, the sample is sent to a controlled atmosphere processing area, where the AT is processed in a grade A isolator.

All materials used for processing are sterile, and packaging sprayed with 70% ethanol or isopropanol before entering the controlled atmosphere processing area. Before entering the grade A isolator, packaging is sprayed with sterile 70% ethanol or isopropanol. Before starting AT processing, decontamination solution (DS) and matrix dissociation solution (MDS) are removed from freezer and warmed to 37°C. In order to speed AT isolation procedure, all solutions are ready to use, prepared beforehand in GMP conditions, including testing for microbial contamination. -

Adipose tissue obtained from liposuction procedure contains variable volume of surgical fluids, which are removed by aspiration into a waste container, after allowing complete phase separation by gravity. Adipose tissue obtained from lipectomy procedure is considerably compact, requiring mechanical mincing into smaller pieces. In a preferred embodiment, mincing can be performed by the use of scissors, blade, scalpel or other sharp instrument into pieces of about 0.5 cm diameter. Adipose tissue sample is further equally fractionated into multiple devices (in even number) for washing (34) and matrix dissociation (35). In a preferred embodiment, the devices are 175 mL conical tubes. The amount of tissue fractionated is weighed and further washed. In a preferred embodiment, washing is performed by the use of warm (37°C) decontamination solution (DS) applied to adipose tissue at 1 :2, 1 : 1 or 2: 1 ratios. In a preferred embodiment, an equal volume of DS compared to adipose tissue (1 :1 ratio) is added to each device with the use of, for example, a pipettor, and the device agitated vigorously. Phase separation by gravity is allowed and DS is further aspirated and discarded. As an example, five washes can be performed. At the last wash, a centrifugation step ensures complete separation of DS from AT. This centrifugation step, is performed at between 100 and 400g, preferably between 200 and 300g, for 3 to 10 minutes cycles, preferably for 5 to 7 minutes cycles. Washing allows removal of surgical fluids, excess of red blood cells (RBC) as well as reduction of any microbial content, frequently present in these tissues due to high contact with donor's skin during collection procedure. In a preferred embodiment, the decontamination solution is one including, but not limited to, vancomycin, gentamicin, cefotaxime in concentrations between 50 and 300 mg_ss/L and amphotericin B deoxycholate in concentrations between 10000 and 30000 UI/L as antibiotic/antifungal agents. In a preferred embodiment, these agents are used in concentration between 1 15-140 mg_ss/L and 13500-16500 UI/L, respectively.

Matrix dissociation solution (MDS) is further added to washed AT, by the use of a pipettor as an example, with the purpose to release the nucleated cells. In a preferred embodiment, MDS is composed of one or more enzymes, basal cell culture media and decontamination solution. Enzymes may include collagenase class I and II, as well as neutral protease at concentration ranging between 0.1 and 0.8 PZ-U/mL, preferably between 0.3 and 0.4 PZ-U/mL. Basal cell culture media may include, but not limited to, aMEM, DMEM, DMEM/Hams F-12, supplemented with 1 to 50%, preferably 15 to 20% of said decontamination solution. MDS is pre-prepared in GMP conditions, tested for sterility and stored at -20°C. Pre-preparation of the MDS allows immediate usage of a validated solution during processing procedure, ultimately reducing time and risk of manipulation errors. Adipose tissue matrix dissociation requires further setting of specific conditions such as temperature and agitation. In a preferred embodiment, devices containing AT and DS at ratios of 1 :1, 2: 1, 1 :2, 3: 1 or 1 :3, preferably at 1 : 1 ratio, are incubated at 37°C for 1 to 120 minutes, preferably 60 minutes, in case of liposuction tissue, or 90 minutes in case of AT collected by lipectomy surgery. During incubation, at temperature between 36 to 38°C, preferably at 36.5 to 37.5°C, devices are under agitation between 20 and 200 rpm, preferably between 100 and 150 rpm. Dissociated tissue (DT) is presented as a yellow suspension, containing only small pieces of white connective tissue. This suspension is further strained through a sterile mesh, into a sterile container, to separate any intact fragments. In a preferred embodiment, DT is strained through a 1 mm a sterile mesh and fractioned into multiple 50 mL conical tubes (in even number), and centrifuged twice for complete separation of nucleated cells from the remaining suspension components (36). Centrifugations are performed between 100 and 400 g, preferably between 200 and 300 g, during 1 to 15 minutes cycles, preferably between 5 and 7 minutes cycles.

Cells are recovered by staged removal of the layered components and further suspended within a refrigerated system (37). In a preferred embodiment, oil is firstly completely aspirated to avoid contact and contamination of cells; then, dissociation solution is partially removed, not to interfere with the cell pellet present at the conical bottom of the tube. The cell pellet is further collected with a pipette into a clean conical tube and suspended to separate any clustered cells. Unclustered cell suspension is further mixed with a cold xeno-free protein solution (CPS) (e.g. 2- 10°C), within a refrigerated system (38), in order to inactivate any remaining enzyme activity. In a preferred embodiment, proteins may include, but not limited to, human serum, human albumin, or recombinant human albumin, at concentration ranging between 0.1 to 40%, preferably between 1% and 20%. Also in a preferred embodiment, the refrigerated system must ensure temperatures within 2 and 10°C, preferably within 4 and 8°C. From this stage further, cell content is maintained refrigerated, preferably within 4-8°C, during all procedures. Purification of the cell suspension takes place to yield a nucleated single cell concentrate, and reduce risk of contamination. By contamination, is understood, tissue debris, cell clusters, red blood cells, residues of DS or MDS. The cell suspension is sequentially filtered (39) through a sterile mesh, with sizes below 130μιη, 90μιη and/or 50μιτ), preferably of 100 μηι and 40 μηι, to yield a suspension of single cells. This is further centrifuged (40) to yield a concentrate, whereas residues of previous solutions (DS and MDS) are discarded through the supernatant. In a preferred embodiment, centrifugations are performed between 100 and 400 g, preferably between 200 and 300 g, during 1 to 15 minutes cycles, preferably between 5 and 7 minutes cycles.

Cell concentrate is suspended in cold xeno-free protein solution) (e.g. 2-10°C), and a red blood cell (RBC) lysis buffer, is added to cell suspension at 1 : 1 , 2:1, 3:1 or 4:1 ratios, preferably at 2:1 ratio, for 1 to 10 minutes, preferably 3 to 5 minutes (41). Reaction is further stopped, preferably by 2x dilution with CPS and immediate centrifugation, preferably between 200 and 300 g, 5 and 7 minutes, to remove haemoglobin released by RBC. Cell concentrate, depleted from RBC is further suspended with CPS and sampled for quality control II (42).

The purified clinical grade cells are sampled for cell counting, viability assessment, immunophenotypic analysis and cell culture. In a preferred method for viability assessment and cell counting, nucleated cells are incubated with trypan blue viability exclusion dye, and counted using a hemacytomer. In a preferred embodiment, clinical grade cell isolation method yields cell number higher than 150000 nucleated cells/gram of adipose tissue, and cell viability higher than 80%.

In a preferred method for immunophenotypic analysis, nucleated cells are incubated with antibodies for specific cell surface markers, including, but not limited to, CD34, CD90, CD105, CD73, CD31, CD45, CD235a, CD44,CD29, CD13, CD10, CD26, CD36, CDl lb, CD49d, CD49e, Cd49f, CD106, CD144, CD133, Cdl46, HLA-ABC and PODXL. In a preferred method for cell culture, nucleated cells are placed in xeno- free cell culture media. A last centrifugation, preferably between 200 and 300 g, 5 and 7 minutes, yields the ready to use clinical grade cell concentrate, and a supernatant. Supernatant is collected for testing towards sterility and absence of mycoplasma and endotoxin (42). Cell concentrate can be transferred to a device suitable for immediate therapeutic application. In a preferred embodiment, cell concentrate is cryopreserved.

The clinical grade cell concentrate defines a cellular product, produced by the defined isolating method, wherein the cell concentrate includes a mixture of sub-populations including preadipocytes, mesenchymal stromal/stem cells (MSC), endothelial cells and endothelial progenitors, pericytes, stromal cells, as well as hematopoietic lineage cells. In a preferred embodiment, isolated clinical grade cells with concomitant expression of CD34+CD45-CD31-, is higher than 30% and concomitant expression of CD34+CD90+ CD 105+CD73+, is higher than 15%.

Cryopreservation solution (CS) (43) is prepared immediately before use, being composed of a protein solution and a cryoprotectant. The preferred protein may include, but not limited to, human serum, human albumin, or recombinant human albumin, at concentration ranging between 0.1 to 40%, preferably between 10% and 20%. The preferred cryoprotectant may include, but not limited to, dimethyl sulfoxide (DMSO), dextran and water, where dimethyl sulfoxide is at concentrations between 40 and 100%, preferably between 45 and 55% and dextran at concentration between 0 and 15%, preferably between 5 and 10%. The cryoprotectant fraction corresponds between 2 and 20% of the cryopreservation solution, preferably between 5 and 10%.

Clinical grade cell concentrate is suspended in cold CS to yield desired cell concentration, ranging from 0.5-10.0 million viable nucleated cells/mL, where the preferred cell density is that ranging from 1.0-2.0 million viable nucleated cells/mL.

The cryopreservation cell suspension is transferred into a cold cryopreservation device (44), barcode labelled with a sample identification code (derived from initial ID code). The preferred cryopreservation device is dependant of the predicted end-use, whereas cryogenic vials or cryogenic bags, both of variable volumes can be chosen. The cold cell-filled cryogenic device is further gradually cooled to cryopreservation temperatures, in a controlled manner (45). In a preferred embodiment, the cold cell- filled cryogenic device is transferred to a controlled rate freezer, where temperature decrease from 4°C to - 40°C is controlled at a 1°C per minute rate and from - 40°C to - 120°C at a - 10°C per minute rate. Devices are immediately transferred to a vapour phase liquid nitrogen tanks and racks properly identified (46).

Cryopreserved cells are placed in a quarantine zone until full quality control (42) assessment is complete and no acceptance criteria have failed. If failed, cryopreserved cells are discarded as biohazard waste and registered as such on the database, for tracking purposes. If acceptance criteria are met (47), the cryogenic device containing the cryopreserved cells is shifted to a final storage position, and registered on the database.

Recovery of cells from cryopreservation is performed immediately before end use

(48) . Thawing of cells is attained by quick immersion of the cryopreservation device in a warm water bath; preferably at 37°C. Thawed cryopreserved cell suspension is further diluted in warm xeno-free protein solution and sampled for quality control III

(49) , including determination of post-thaw viable cell number, cell viability and immunophenotypic expression. In a preferred embodiment, xeno-free protein solution may include, but not limited to, human serum, human albumin, or recombinant human albumin, at concentration ranging between 0.1 to 40%, preferably between 10% and 20%.

Acceptance criteria include values not inferior than 30% of those obtained before cryopreservation, ideally not inferior than 10%. If criteria are met, clinical grade cells may be released for therapeutic application (50).

Testing for microbial contamination is performed, by using standard systems. In a preferred embodiment, an automated microbial detection system is used. Specific testing is performed for compliance with regulatory agencies and guidelines. Samples are inoculated into aerobic and anaerobic microorganism growing media, or other if necessary, and stored at room temperature before incubation at specific temperature. It is mandatory that a sterility testing laboratory is physically separated from tissue manipulation facilities to avoid cross-contamination. All sterility testing cultures are incubated at an appropriate temperature, for a minimum of 14 days, with periodic inspection and registry of results on a dedicated form. Absence of turbidity or colony formation and/or undetection of carbon dioxide production is an indicator of the absence of microbial contamination.

Examples

Clinical grade cells obtained from adipose tissue through the systems, methods and devices are sampled for quality control at distinct stages. The following examples demonstrate qualitative and quantitative data regarding cell purity, cell yield, cell viability and cell immunophenotype obtained by the preferred embodiments of the present invention, and how these outperform systems of the prior art.

Materials and Methods

Human clinical grade cells were isolated from adipose tissue as follows:

A quality management system was set, together with a database, to ensure traceability of all information related to a specific adipose tissue sample, in accordance to guidelines and regulations applicable to transplantation units and medicinal products.

A transportation unit was assembled beforehand, containing a sterile adipose tissue collection device, a temperature logger, a blood collection kit, cold accumulators, documentation and labels for identification of all documents and devices with an ID code and a barcode. The transportation unit was sent to an authorized adipose tissue collection entity (clinic or hospital). Human adipose tissue was harvested by lipectomy, approximately 400 g, and stored in the collection device, all in aseptic conditions. To minimize any residual microbial contamination, a 1 : 1 ratio of decontamination solution is added. Collection device was further packed in transportation unit, together with collected blood tubes and documentation, and sealed for transportation to clean room, GMP processing facilities.

At processing facilities, quality control (QC) was performed by inspection of the transportation unit, and registry on a standardized form, the following: a) confirmation of the transportation unit expiry date was after the unit's arrival date; b) confirmation of the presence and integrity of security seal; c) integrity of the shipping container; d) confirmation of collection device with AT within security bag with absorbent - annotation of volume collected and signs of leakage; e) confirmation of blood tubes within security bag with absorbent; f) confirmation of barcode label on the collection device with AT and blood tubes; g) the temperature logger has registered temperature values within the range 2°C-8°C, between cooling period and reception; h) confirmation that the AT was collected within the past 48h; i) confirmation of documentation and complete registry of information and g) confirmation of packaging performed as instructed. Given that conditions were met, sample followed to the controlled atmosphere processing area, being the AT processed in a grade A isolator.

Before starting AT processing, decontamination solution (DS) and matrix dissociation solution (MDS) (pre-prepared) were removed from freezer and warmed to 37°C. Adipose tissue obtained from lipectomy procedure was further minced, by the use of sterile scissors, into pieces of about 0.5 cm diameter. Adipose tissue sample was further equally fractionated into multiple devices (in even number) such as 175 mL conical tubes. The amount of tissue fractionated was weighed and further washed by the use of warm (37°C) decontamination solution (DS). Equal volume as that of adipose tissue (1 : 1 ratio) was added to each device with the use of a pipettor, and the device agitated vigorously. Phase separation by gravity was allowed and DS further aspirated and discarded. This was repeated five times. At the last wash, a centrifugation step ensured complete separation of DS from AT (200 g, 5 minutes). Washed AT was further inoculated, by the use of a pipettor, with equal volume of matrix dissociation solution (MDS) and incubated at 37°C for 90 minutes, with agitation at 125 rpm. The dissociated tissue (DT) was further strained through a sterile lmm mesh, into a sterile container, to separate any intact fragments, and further fractioned into multiple 50 mL conical tubes (in even number). Centrifugation was performed twice (300g 5 minutes, shake tubes, 300g 10 minutes).

Cells were further recovered by staged removal of the layered components: oil was firstly completely aspirated to avoid contact with cells; then, dissociation solution was partially removed, not to interfere with the cell pellet present at the conical bottom of the tube. The cell pellet was further collected with a pipette into a clean conical tube and suspended to separate any clustered cells. Unclustered cell suspension was further mixed with a cold xeno-free protein solution (CPS), within a refrigerated system. From this stage further, cell content was maintained refrigerated, during all subsequent steps.

Purification of the cell suspension took place to yield a nucleated single cell concentrate, and reduce risk of contamination by tissue debris, cell clusters, red blood cells, residues of DS or MDS. The cell suspension was sequentially filtered through 100 μηι and 40 μηι sterile mesh, to yield a suspension of single cells. This was further centrifuged (300g 5 minutes) to yield a cell concentrate, whereas residues of previous solutions (DS and MDS) are discarded through the supernatant. Cell concentrate was suspended in cold xeno-free protein solution, and inoculated with a red blood cell (RBC) lysis buffer, at a 1 :2 ratio for 3 minutes. Reaction was further stopped by 2x dilution with CPS and immediate centrifugation, to remove haemoglobin released by RBC. Cell concentrate, depleted from RBC was further suspended with CPS and sampled for quality control II, such as cell counting, viability assessment, immunophenotypic analysis and cell culture.

A last centrifugation yields the ready to use clinical grade cell concentrate, and a supernatant. Supernatant was collected for sterility, mycoplasma and endotoxin testing and cell concentrate was prepared for cryopreservation.

Cryopreservation solution (CS) was prepared immediately before use, being composed of human albumin and 10% dimethyl sulfoxide (DMSO)/ dextran (55%/5%). Clinical grade cell concentrate was suspended in cold CS to yield a cell concentration, of 1.3 million viable nucleated cells/mL. Cryopreservation cell suspension was transferred into a cold 1.5 mL cryopreservation device, barcode labelled with sample identification code (derived from initial ID code). Cold cell-filled cryogenic device is further gradually cooled to cryopreservation temperatures, in a controlled rate freezer, where temperature decrease from 4°C to - 40°C was controlled at a 1°C per minute rate and from - 40°C to - 120°C at a - 10°C per minute rate. Devices were immediately transferred to a vapour phase liquid nitrogen tanks and racks properly identified.

Cryopreserved cells were placed in a quarantine zone until full quality control assessment was complete and no acceptance criteria have failed. At this stage, sample was shifted to final storage position, and registered at database.

Recovery of cells from cryopreservation was performed for quality control. Thawing of cells was attained by quick immersion of cryopreservation device in a 37°C water bath. Cell suspension was further diluted in warm xeno-free protein solution (10% human albumin) and sampled for post-thaw viable cell number, cell viability and immunophenotypic expression.

Testing for microbial contamination is performed, by an automated microbial detection system (BactAlert). Specific testing was performed for compliance with regulatory agencies and guidelines. Samples were inoculated into aerobic and anaerobic microorganism growing media, and stored at room temperature before incubation at specific temperature for a minimum of 14 days. Undetection of carbon dioxide production is an indicator of absence of microbial contamination.

As for cell viability assessment and cell counting, nucleated cells are incubated with trypan blue viability exclusion dye, and counted using a hemacytomer and microscope. Simultaneously, assessment for contaminants, such as oil droplets, cell aggregates, tissue debris and red blood cells was performed through visual inspection. Automated cell count systems were not used, or recommended, given the high probability of false positive outcomes, as result of our own experience, as well as reported in prior art.

As for immunophenotyping, surface marker (mesenchymal, hematopoietic and endothelial) expression was evaluated by incubating cell suspensions with fluorescent labelled antibodies followed by data acquisition and analysis on a FACSCanto cytometer and the resulting data processed using FACSDiva software. Characterization was performed by the analysis of concomitant expression of four surface markers, providing highly rigorous outcomes of the sub-populations within the isolated cells. This approach is not commonly reported in prior art, where individual markers are usually analysed.

Evaluation was performed in the following groups: purification state of isolated cells (unpurified or purified), protein source used for cryopreservation (human or animal), procedure step (after isolation or after cryopreservation) and harvesting technique (liposuction or lipectomy).

Results

As observed in figure 4, the purification steps yield a cell suspension visibly purified, free from oil droplets (a), cell aggregates / tissue debris (b) and red blood cells (c). Quantitatively, as observed in figure 5, the purification steps yield a significant reduction in the amount of red blood cells (95%±4), as compared to those present in unpurified cells, both of these significantly lower than number of RBC present in peripheral blood. Given this, the method yields a cell population for therapeutic application containing residual amounts of the mentioned contaminants, which ultimately:

1. Improve characterization of the cell fraction - by minimizing interference of contaminants, data obtained for quantification of cell parameters such as cell number, cell viability and cell immunophenotype are much more reliable;

2. Reduce risk associated with the presence of these contaminants, when used in therapeutic applications.

As for the before mentioned cell characterization parameters, it is observed in figure 6 that the purification steps do not interfere with the end product cell yield or cell viability of the isolated cells, given that no significant difference was observed between purified and unpurified groups: both revealed an extraordinary cell viability 84%±6 and 84%±5, respectively, and cell yield 0.25±0.1 1 and 0.28±0.08 million cells/g adipose tissue, respectively, above those described by other cell isolation systems described in the prior art. As for cryopreservation system (figure 8), no impact on cell viability was determined by the use of xeno-free cryopreservation solution, as compared to well-established animal serum (86%±3 vs 83%±5, respectively). As for the cryopreservation and thawing system as a whole (figure 10), no prejudicial effect of the preferred embodiment was quantified over initial, pre- cryopreservation viability of the cells (89%±5 vs 84%±6, respectively).

Concomitance of mesenchymal cell surface markers (CD73, CD90 and CD 105) was evaluated in the presence and absence of CD34. In the presence of CD34, no differences were observed on the expression of the markers for all the groups described. In the absence of CD34, a significant difference was seen when comparing the groups regarding purification sate of the isolated cells, in what concerns the expression of CD34-CD90+, with a higher expression in the purified group. Evaluation of such markers in the groups comparing the harvesting technique, a significant difference was observed in the subpopulation CD34- CD73+CD90+CD105+, with a higher expression when the tissue is harvested by lipectomy.

Subpopulations identified as endothelial lineage were also evaluated. Concerning these subpopulations, in the purification and procedure steps differences were observed for the subpopulation CD31+CD34+CD45-.with higher expression in the purified group and cryopreservation. Moreover in procedure step, differences were seen for the subpopulation CD34+CD45-, with higher expression in the cells after cryopreservation.

Claims

Claims

1. A method of isolating cells from adipose tissue, the method comprising:

a) providing a sample of adipose tissue;

b) optionally, evaluating the sample to ensure it complies with pre-determined criteria;

c) optionally, mincing the sample into smaller pieces;

d) washing the sample with a decontamination agent;

e) dissociating the matrix of the cells of the sample using a matrix dissociating agent;

f) isolating the cells;

g) purifying the cells; and

h) optionally, evaluating the cells of the sample to ensure they comply with predetermined criteria,

wherein the method is carried out such that no non-human biological components are introduced into the sample, and wherein isolated cells are produced.

2. The method of claim 1, further comprising xeno-free cryopreservation of the isolated cells.

3. The method of claim 1 or claim 2, wherein washing the sample with a decontamination agent sterilises the sample.

4. The method of any preceding claim, wherein the decontamination agent is a solution comprising one or more agents selected from vancomycin, gentamicin, cefotaxime, and amphotericin B deoxycholate.

5. The method of any preceding claim, wherein the matrix dissociating agent is a solution containing an enzyme which breaks down the cell matrix.

6. The method of claim 5, wherein the enzyme is selected from collagenase class I and II, and neutral proteases.

7. The method of any preceding claim, wherein the matrix dissociating agent comprises basal cell culture media.

8. The method of claim 7, wherein the matrix dissociating agent comprises a decontamination agent.

9. The method of any preceding claim, wherein the matrix dissociation step comprises incubation of the sample with the matrix dissociating agent.

10. The method of claim 9, wherein the sample is incubated with the matrix dissociating agent at 30 to 38°C, preferably at 36 to 37°C.

11. The method of claim 9, wherein the sample is incubated with the matrix dissociating agent, for between 15 minutes and 120 minutes, preferably between 45 minutes and 90 minutes.

12. The method of any preceding claim, wherein the matrix dissociation step comprises agitating the sample when in contact with the matrix dissociation agent.

13. The method of claim 12, wherein the sample is agitated with the matrix dissociation agent at between 20 and 200 rpm, preferably between 100 and 150 rpm.

14. The method of any preceding claim, wherein the cell isolation step comprises straining the sample through a mesh to separate any intact fragments.

15. The method of any preceding claim, wherein the cell isolation step comprises centrifuging the dissociated cells to separate the cells from any other remaining components.

16. The method of any preceding claim, wherein the cell isolation step comprises centrifuging the cells and resuspending the cells in xeno-free protein solution.

17. The method of claim 16, wherein the xeno-free protein solution is at a temperature between 2-10°C, preferably 4-8°C.

18. The method of any preceding claim, wherein the cell isolation step comprises cooling the cells to a temperature between 2-10°C.

19. The method of claim 18, wherein the cells are maintained at 2-10°C for all subsequent steps in the method until cryopreservation, if carried out.

20. The method of any preceding claim, wherein the cell purification step comprises sequentially filtering the cell suspension using different mesh size filters.

21. The method of any preceding claim, wherein the cell purification step comprises removing red blood cells from the sample.

22. The method of claim 21, wherein the red blood cells are removed from the sample using a red blood cell (RBC) lysis buffer and subsequently centrifuging the sample to remove haemoglobin released by the red blood cells.

23. The method of any preceding claim, wherein the isolated cells produced by the method have a cell number of higher than 150,000 nucleated cells/gram of adipose tissue.

24. The method of any preceding claim, wherein the isolated cells produced by the method have a cell viability of higher than 80%.

25. The method of any preceding claim, wherein the isolated cells produced by the method have concomitant expression of CD34+CD45-CD31- of higher than 30%.

26. The method of any preceding claim, wherein the isolated cells produced by the method have concomitant expression of CD34+CD90+CD105+ CD73+ of higher than 15%.

27. A xeno-free method of isolating cells from adipose tissue according to any preceding claim, the method comprising:

a) providing a sample of adipose tissue;

b) optionally, mincing the sample into smaller pieces;

c) washing the sample with a decontamination agent to sterilise the sample, wherein the decontamination agent is a solution comprising one or more agents selected from vancomycin, gentamicin, cefotaxime, and amphotericin B deoxycholate; d) dissociating the matrix of the cells of the sample by incubating the sample with a matrix dissociating agent at 36 to 37°C for between 45 minutes and 90 minutes, and agitating the sample at between 100 and 150 rpm, wherein the matrix dissociating agent is a solution containing an enzyme selected from collagenase class I and II, and neutral proteases;

e) straining the sample through a mesh to separate any intact fragments;

f) centrifuging the dissociated cells to separate the cells from any other remaining components;

g) centrifuging the cells and resuspending the cells in a xeno-free protein solution, wherein the xeno-free protein solution is at a temperature between 2-10°C;

h) cooling the cells to 2-10°C and maintained the cells at 2-10°C for the subsequent steps in the method;

i) sequentially filtering the cells using different mesh size filters; and

j) removing red blood cells from the sample using a red blood cell (RBC) lysis buffer and subsequently centrifuging the sample to remove haemoglobin released by the red blood cells,

wherein the method is carried out such that no non-human biological components are introduced into the sample, and wherein isolated clinical grade regenerative cells are produced which have a cell number of higher than 150,000 nucleated cells/gram of adipose tissue and a cell viability of higher than 80%.

28. Isolated cells produced by the method of any preceding claim.

29. The isolated cells of claim 28 for use in therapy.