WO2012070001A1 - Technique de culture d'explant pour l'isolement de cellules souches mésenchymateuses à partir de tissu adipeux - Google Patents

Technique de culture d'explant pour l'isolement de cellules souches mésenchymateuses à partir de tissu adipeux Download PDF

Info

Publication number
WO2012070001A1
WO2012070001A1 PCT/IB2011/055252 IB2011055252W WO2012070001A1 WO 2012070001 A1 WO2012070001 A1 WO 2012070001A1 IB 2011055252 W IB2011055252 W IB 2011055252W WO 2012070001 A1 WO2012070001 A1 WO 2012070001A1
Authority
WO
WIPO (PCT)
Prior art keywords
media
asc
tissue
explant
concentration ranging
Prior art date
Application number
PCT/IB2011/055252
Other languages
English (en)
Inventor
Swathi Sundar Raj
Nancy Priya
Original Assignee
Stempeutics Research Pvt. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stempeutics Research Pvt. Ltd. filed Critical Stempeutics Research Pvt. Ltd.
Publication of WO2012070001A1 publication Critical patent/WO2012070001A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • the present disclosure relates to the field of stem cells in general, while in particular it relates to the isolation of mesenchymal stem/stromal cells (MSC) from adipose tissue by explant culture method.
  • MSC mesenchymal stem/stromal cells
  • the method provides stress-free, cost effective isolation of enriched mesenchymal stem cells for further expansion and use in clinical/therapeutic applications.
  • MSC Mesenchymal stem/stromal cells
  • MSC myelogenous leukemia
  • Wharton's jelly adipose tissue
  • umbilical cord blood synovium
  • skeletal muscle dental pulp
  • limbal tissue amniotic fluid
  • placenta and menstrual blood a particularly advantageous source as it can be obtained in much larger quantity when compared to other tissues, has a very high MSC content, and can be easily harvested by liposuction.
  • Adipose tissue-derived MSC are in fact being evaluated in clinical trials for several pathologies including ischemic heart disease, Crohn's disease, liver cirrhosis, limb ischemia and diabetes, as well as in a variety of cosmetic and reconstructive procedures.
  • Adipose tissue is composed of several cell types including mature adipocytes, preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells and their progenitors, resident monocytes/macrophages, lymphocytes, and the multipotent stem/stromal cells.
  • the Conventional method for isolation of MSC from adipose tissue universally involves dissociation of the adipose tissue to release the different cell populations by chemical (enzymatic, collagenase treatment) or physical (ultrasonic) methods, or a combination of both.
  • tissue dissociation is defined as the process of obtaining single cell suspensions from primary tissue by breaking down the tissue extracellular matrix. Such methods typically place considerable stress on the cells due to enzyme treatment and time spent (3-4 hours) outside the physiological environment (37°C, 5% C0 2 ).
  • ASC have recently been shown to be composed of several subpopulations that differ in surface marker expression and exhibit distinct differentiation potentials [Rada, T., et al (201 1). Stem Cell Reviews, 7:64-76 and Paredes, B et al. (2010). Journal of Tissue Engineering and Regenerative Medicine, 10.1002/term.351]. Enzymatic digestion followed by centrifugation is also a time consuming and stressful process.
  • the PCT application WO/2008/129563 relates to a process of isolation of MSC mainly from bone marrow and culture of the stem cells. This application does not specifically disclose the method of isolation of MSC from Adipose tissue but makes a statement that MSC can be isolated from various other sources including adipose tissue.
  • Liposuction used in present disclosure on the other hand is a minimally invasive procedure, and hence the preferred procedure for fat harvesting for stem cell isolation.
  • the immunogenicity and immunomodulatory nature of cells derived from fat tissue explants has also not been determined in the report by Jing et.al. As adipose tissue is composed of several cell types described earlier, it is not known if the cells obtained by explant culture would have immune privilege, which is absolutely essential for clinical utility of the ASC.
  • Adipose tissue-derived Mesenchymal Stem Cells in which ASC can be obtained without subjecting the cells to the stress-strain as well as all the above mentioned disadvantages associated with the normal conventional method of isolation.
  • Present disclosure discloses the utility of the explant culture system for derivation of ASC from adipose tissue. The method was evaluated not only with adipose tissue obtained by surgical resection, but also with tissue obtained by liposuction, which is minimally invasive and hence the preferred procedure for fat harvesting.
  • the present disclosure relates to a method of isolating mesenchymal stem cells from adipose tissue non-enzymatically, said method comprising acts of a) harvesting adipose tissue, b) washing the harvested adipose tissue with buffer solution and separating fat tissue from the buffer solution, c) mincing the separated fat tissue to obtain tissue explants and d) adding pre -warmed culture media over the tissue explants and incubating to isolate the mesenchymal stem cells non-enzymatically; a media for isolating mesenchymal stem cells from adipose tissue non-enzymatically, said media comprising basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a-MEM), supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-di
  • DMEM Dulbecco's
  • Fig. 1 Shows isolation of ASC by explant culture: A, adipose tissue explants; B, C, migration of fibroblastic cells from the tissue explants on day 3. D, E, explant derived cells on day 6; F, G, explant derived cells on day 9. B, D and F represent lipoaspirate tissue explants; C, E and G represent resected tissue explants. Scale bars represent a magnification of 100 microns.
  • Fig. 2 Shows comparison graphs and photos of Growth kinetics of ASC derived by explant culture and collagenase digestion methods.
  • A fold expansion from P0 to P10 for explant (lipoaspirate and resected tissue) and collagenase-derived ASC;
  • B cumulative population doubling at each passage for explant (lipoaspirate and resected tissue) and collagenase-derived ASC from P0 to P10;
  • C mean doubling time for explant (lipoaspirate and resected tissue) and collagenase-derived ASC over 10 passages.
  • Ex-L lipoaspirate explant-derived ASC;
  • Ex-T resected tissue explant-derived ASC; Coll., collagenase-derived ASC.
  • Data is represented as mean ⁇ standard deviation for at least 4 biological replicates for each condition.
  • D senescence associated ⁇ -galactosidase staining for ASC derived by explant and enzymatic techniques at P20.
  • Micrographs show a representative set of images from 4 biological replicates for each condition, scale bars represent a magnification of 50 microns.
  • Fig. 3 Shows comparison of detection of cell surface markers expressed by explant and collagenase-derived ASC at P4 by immunophenotyping and flow cytometric analysis. Open histograms represent staining with isotype control antibodies, shaded histograms represent staining with the specific antibody. The percentage positive cells for each marker is calculated after subtraction of the non-specific fluorescence obtained with the isotype control antibodies. Data shows a representative set of histograms from 4 biological replicates for each condition. Coll- ASC, collagenase derived ASC.
  • Fig. 4 Shows comparison of multipotentiality of explant and collagenase-derived ASC.
  • A, B oil-red O staining of adipogenically induced ASC isolated by explant culture (A) and the corresponding collagenase control (B).
  • C the stain from both uninduced and induced cultures was extracted using chloroform and methanol (2: 1) and quantified by spectrophotometry.
  • D, E alizarin red S staining of osteogenically induced ASC isolated by explant culture (D) and the corresponding collagenase control (E).
  • F the stain from both uninduced and induced cultures was extracted using acetic acid and ammonium hydroxide and colorimetrically estimated.
  • G H
  • H alcian blue staining of induced chondrocyte pellets of ASC isolated by explant culture (G) and the corresponding collagenase control (H).
  • I semi-quantitative RT-PCR analysis for expression of chondrogenic genes in induced pellets of explant-derived ASC (Ex.) and the corresponding collagenase control (Coll.). 18s ribosomal R A expression was used to normalize cDNA concentration for each sample set.
  • Data depicts gene expression in two pairs of samples out of 4 pairs of biological replicates analyzed. Micrographs show a representative set of images from 4 biological replicates for each condition, scale bars represent a magnification of 50 microns. Graphs represent mean ⁇ standard deviation from 4 biological replicates for each condition.
  • Fig. 5 shows graphical representation of Immunogenicity and immunosuppressive capacity of explant-derived ASC.
  • A mitomycin-C arrested ASC fail to stimulate proliferation of allogeneic PBMC. Mismatched PBMC (allo-PBMC) were used as positive controls for PBMC stimulation at the corresponding stimulator to responder ratios.
  • B inhibition of mixed lymphocyte reaction by mitomycin-C arrested ASC. Data is represented as mean ⁇ standard deviation for 4 biological replicates for each condition.
  • the present disclosure relates to a method of isolating mesenchymal stem cells from adipose tissue non-enzymatically, said method comprising acts of:
  • the present disclosure also relates to a media for isolating mesenchymal stem cells from adipose tissue non-enzymatically, said media comprising basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a- MEM), supplemented with Serum, growth factor, Sphingosine-1- Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.
  • basal medium selected from group comprising Dulbecco's Modified Eagle Medium (DMEM) or a - Minimum Essential Medium (a- MEM), supplemented with Serum, growth factor, Sphingosine-1- Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.
  • DMEM Dulbecco's Modified Eagle Medium
  • a- MEM Minimum Essential Medium
  • the harvesting is carried out using liposuction, lipectomy, lipoaspiration and resection or any combination thereof.
  • the buffer solution is selected from group comprising normal saline, Phosphate Buffered Saline and basal medium; preferably Phosphate Buffered Saline.
  • the media comprises of basal medium selected from group comprising DMEM or a-MEM, supplemented with Serum, growth factor, Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotic or any combination thereof.
  • the Serum is selected from group comprising Human serum (HS), Fetal Bovine Serum (FBS), Human Plasma (HP) and Human Platelet Lysate (HPL).
  • HS Human serum
  • FBS Fetal Bovine Serum
  • HP Human Plasma
  • HPL Human Platelet Lysate
  • the DMEM is selected from group comprising DMEM-KO, DMEM-LG, DMEM-HG and DMEM-F12, each having concentration ranging from about 80% to about 99%.
  • the a-MEM is having concentration ranging from about 80% to about 99%.
  • the Serum is having concentration ranging from about 1% to about 20%, preferably about 10%.
  • the (HP) is having concentration ranging from about 1 to about 20% and the (HPL) is having concentration ranging from about 1% to about 20%.
  • the growth factor is selected from group comprising basic Fibroblast Growth Factor (bFGF) and Platelet Derived Growth Factor (PDGF) or combination thereof, each having concentration ranging from about 1 ng/ml to about 20 ng/ml.
  • bFGF basic Fibroblast Growth Factor
  • PDGF Platelet Derived Growth Factor
  • the Sphingosine-1 -Phosphate is having concentration ranging from about 0.01 ⁇ to about 5 ⁇ .
  • the Glutamine or Glutamine- dipeptide is having concentration ranging from about 50 mM to about 500 mM, preferably about 200 mM.
  • the antibiotic is selected from group comprising Penicillin having concentration ranging from about 50 to about 200 U/ml, preferably about 100 U/ml; Streptomycin having concentration ranging from about 50 to about 200 ⁇ g/ml, preferably about 100 ⁇ g/ml; and Gentamycin having concentration ranging from about 10 ⁇ g/ml to about 200 ⁇ g/ml, preferably about 25 ⁇ g/ml.
  • the incubation is carried out in humidified 5% C0 2 incubator at about 37°C, for time duration ranging from about 4 days to about 20 days and sub-cultured for duration ranging from about 7 days to about 10 days.
  • the media is used in amount ranging from about 1ml to about 5ml, preferably about 2.5 ml.
  • the present disclosure relates to a kit comprising media components as above along with instructions for preparation of the media.
  • the present disclosure also relates to a method of assembling a kit as above, said method comprising act of combining components as above to arrive at the kit.
  • the buffer solution is selected from a group comprising normal saline, phosphate buffered saline (PBS) and basal medium in order to remove blood.
  • the completed culture media comprises basal medium and supplements to support cell growth.
  • Basal medium is selected from DMEM or a-MEM, supplemented with bovine or human serum, or plasma or human platelet lysate, growth factors; Sphingosine-1 -Phosphate, Glutamine or glutamine-dipeptide and antibiotics, and the explant tissues are cultured in complete culture media.
  • the instant disclosure presents a media composition used for culturing the MSC isolated by explants method/non-enzymatic method.
  • the MSC are isolated without the use of any enzymes for tissue dissociation unlike the conventional method wherein enzymes like collagenase are used, hence it is also referred to as non-enzymatic method.
  • the instant disclosure describes a novel, gentle, much simplified and cost-effective technique for isolation of adipose tissue-derived MSC (ASC) by explant culture, without using conventionally known tissue dissociation methods.
  • ASC tissue-derived MSC
  • the present disclosure demonstrates that MSC are isolated from undissociated adipose tissue.
  • Explant culture' refers to transferring intact tissue obtained by lipectomy/liposuction into a suitable culture medium.
  • the tissue is not dissociated or processed using chemical or physical means, and thus places minimal stress on the cells.
  • the processing time is also very rapid, as the surgically removed tissue is transferred to a physiological environment (37°C, 5% C0 2 , isotonic culture medium containing relevant growth factors) with minimal time delay (30-60 min).
  • This instant method also eliminates use of enzymes such as collagenase, hence also referred to as non-enzymatic method, which would dramatically reduce cost of processing, as well as negate problems surrounding use of xeno-derived or bacterial-derived agents for isolating cells intended for clinical use in humans.
  • Another advantage is that the amount of tissue/lipoaspirate required for explant culture is also much lesser than the amount of tissue/lipoaspirate required for isolating cells by enzymatic means. This technique yields a population of stromal cells that is highly enriched for MSC.
  • ASC isolated and cultured as described by explant culture method of present disclosure showed typical spindle shaped morphology characteristic of MSC.
  • the explant-derived ASC show potent immunosuppressive capacity in a dose-dependent manner and efficiently differentiate towards adipogenic, osteogenic and chondrogenic cell lineages.
  • the tissue used in the instant disclosure is Adipose tissue obtained by any known medical procedure in art but not limiting to methods such as liposuction, lipoaspiration and lipectomy.
  • the composition of the culture medium comprises of Basal medium selected from Dulbecco's Modified Eagle Medium (DMEM) or Alpha-Minimum essential medium (a-MEM).
  • DMEM is selected from a group comprising DMEM-KO, DMEM-LG, DMEM-HG, DMEM-F12 (1 : 1); basal medium is supplemented with 1 to 20% Fetal Bovine Serum (FBS) or 1 to 20% Human Serum (HS) or 1 to 20% Human Plasma or 1 to 20% Human Platelet Lysate (HPL); growth factors such as Fibroblast Growth Factor (bFGF) or Platelet Derived Growth Factor (PDGF), Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotics.
  • FBS Fetal Bovine Serum
  • HS Human Serum
  • HPL Human Platelet Lysate
  • growth factors such as Fibroblast Growth Factor (bFGF) or Platelet Derived Growth Factor (PDGF), Sphingosine-1 -Phosphate, Glutamine or Glutamine-dipeptide and antibiotics.
  • bFGF Fibroblast Growth Factor
  • explant culture technique can also be used for isolation of ASC from animal fat tissue, for applications in veterinary regenerative medicine.
  • explant culture technique can also be used for isolation of ASC from animal fat tissue, for applications in veterinary regenerative medicine.
  • Example 1 Method of isolation of Mesenchymal Stem Cells by Explant culture
  • adipose tissue obtained by Lipoaspirate (-10 ml) or resected tissue ( ⁇ 5g) is washed with Saline or Phosphate Buffered Saline (PBS) to remove blood.
  • Lipoaspirated tissue is preferably used as liposuction is a less invasive method of fat harvest compared to the surgical method, and less tedious for ASC isolation when compared to resected tissue.
  • Fat tissues can be lipoaspirated from mammal for explants culture.
  • the tissue is washed by mixing it with an equal a m o u n t o f buffer in a sterile tube and resting it for 2-5 min, t h u s allowing the aqueous buffer fraction containing blood to separate below the fat fraction.
  • the buffer is then removed by aspiration.
  • a small quantity of the fat tissue ( ⁇ 1 g for a 100 mm culture dish) is then transferred to a sterile culture dish and minced into smaller fragments of about 2-10 mm 3 ; preferably 5mm 3 with the help of sterile forceps.
  • the figure la shows the fragments of adipose tissue, henceforth referred to as tissue explants, evenly distributed over the surface of the culture dish. Approximately 0.5-2 g preferably lg of tissue was plated per 100 mm dish. The explants are cultured in Knock out Dulbecco's medium (DMEM-KO) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2mM glutamine and antibiotic (Invitrogen). Minimal quantity of about 1-5 ml, preferably 2.5 ml of pre-warmed (37°C) culture medium is then added over the tissue explants such that the explants still remain in contact with the surface of the culture dish.
  • DMEM-KO Knock out Dulbecco's medium
  • FBS fetal bovine serum
  • culture media means one and the same.
  • the complete media comprises of a basal medium supplement with serum, and other growth factor which support the cell expansion.
  • Serum supplement FBS when replaced with HS, HPL or HS it is referred to as xeno-free culture condition.
  • 'Explant culture' refers to transferring intact tissue obtained by lipectomy/liposuction/lipoaspiration (fig. lb, d, f) and by surgical resection (fig. lc, e, and g) into a suitable culture medium thus placing minimal stress on the cells.
  • the dish containing the explants is then transferred into a humidified incubator where it is maintained at 37°C, 5% C0 2 for a period of 4-10 days during which it is monitored regularly for outgrowth of MSC from the explants.
  • the explants tissue is then removed from the dish and the outgrown MSC are further cultured for 7-10 days in fresh culture medium until a confluent monolayer is obtained.
  • These initial cells referred to as passage 0 (P0), were further sub-cultured at a seeding density of 2000 cells /cm 2 and serially passaged until they reached replicative senescence.
  • Figure If and lg show the cell yield obtained at confluence (passage 0), which is in the range of 5-8 xlO 5 cells /g of initial explant tissue plated.
  • Figure lb, d, and f show the explant culture performed using adipose tissue obtained by liposuction and Figure lc, e, g by surgical resection.
  • Figure lb, c shows spindle-shaped cells migrating out of both types of explants (liposuction & surgical resection) onto the culture dish within 3-4 days of culture.
  • Figure Id and figure le depicts that the migrated cells possess typical mesenchymal morphology and continue to proliferate after removal of the explant tissue.
  • Explant method of present disclosure yields a pure and enriched population of ASC that are capable of self renewal, multilineage differentiation and immunosuppression. They are also hypoimmunogenic and hence suitable for both autologous and allogenic cell therapy upon further expansion.
  • this method can be used to obtain clinical grade ASC from adipose tissue by using a xeno-free culture condition comprising of basal medium supplemented with Human Serum or Human Platelet Lysate(HPL) or serum-free basal medium supplemented with growth factors and sphingosine-1 -phosphate in culture medium instead of using FBS.
  • a xeno-free culture condition comprising of basal medium supplemented with Human Serum or Human Platelet Lysate(HPL) or serum-free basal medium supplemented with growth factors and sphingosine-1 -phosphate in culture medium instead of using FBS.
  • the Xenofree culture conditions comprises of using basal medium plus human serum or HPL instead of FBS.
  • the ASC can be cultured in a Serum- free conditions comprising of serum-free media (proprietary) such as Mesencult-XF and Stempro MSC-SFM supplemented with growth factor.
  • serum-free media proprietary
  • culturing in xeno-free or serum free medium will make the complete process xeno/serum-free, as compared to enzymatic digestion where the enzyme is typically of bacterial origin.
  • the cells isolated by the explant technique are similar to cells obtained by enzymatic digestion in terms of their physical and biological characteristics while having several advantages when compared to enzymatic method.
  • the ASC obtained by explants method is checked for growth kinetics and senescence against ASC obtained by enzymatic (collagenase) method.
  • the lipoaspirates taken for explant culture were also simultaneously processed enzymatically by collagenase digestion, and the SVF obtained was seeded at a density of
  • explant-derived ASC 10 cells/cm to obtain passage 0 (P0) cells(see Fiure2).
  • the growth characteristics of explant-derived ASC is then validated by comparing against the corresponding ASC samples isolated by collagenase digestion, cultured under identical conditions from P0 onwards.
  • Both explant and collagenase derived ASC showed very similar proliferation kinetics as observed in their rate of expansion (Fig. 2a), cumulative population doublings (Fig. 2b) and doubling time (Fig. 2c). No difference is observed in the growth parameters of cells obtained from lipoaspirate and resected tissue explants.
  • Both explant and collagenase-derived ASC can be maintained in culture for up to 20 passages, corresponding to an expansion range of 10 10 -10 15 fold over 42-52 population doublings, beyond which their proliferation declined.
  • Values represent mean percentage positive cells ⁇ standard deviation.
  • the flow cytometric analysis of cell surface markers for explant and collagenase-derived ASC is presented in Table 1.
  • the percentage of CD34+ cells for collagenase-derived ASC ranged between 14-16 % (average: 15%) at P0 and 0.5-5.6 % (average: 3%) at PI .
  • explant-derived ASC are enriched for CD90, CD44 and CD73
  • CD80 and CD86 (Fig. 3). No difference is observed in mesenchymal, hematopoietic or
  • Values represent mean percentage positive cells ⁇ standard deviation.
  • ASC isolated by explant and collagenase method is mitotically arrested and cultured with naive allogeneic peripheral blood mononuclear cells (PBMC) at different stimulator:
  • PBMC peripheral blood mononuclear cells
  • responder ratios As positive control for lymphocyte stimulation, mismatched PBMC is used as stimulator cells at corresponding stimulator: responder ratios. Both preparations of ASC did not elicit a proliferative response in T cells of the PBMC at all ratios tested
  • the immunosuppressive properties of ASC isolated by explant culture and collagenase digestion are compared by testing their ability to suppress a mixed lymphocyte reaction
  • MLR MLRs is established using PBMC from mismatched donors, and cultured either in the presence or absence of growth-arrested ASC at different MSC:MLR ratios.
  • Lymphocyte proliferation is strongly suppressed in the presence of ASC, and this inhibition is found to be dose dependent (Fig. 5b). Explant-derived ASC is thus found to be equivalent to collagenase-derived ASC in their immunosuppressive potency.
  • Adipocyte differentiation of both ASC is detected by staining of accumulated lipid vacuoles with oil red O (Fig. 4a, b). The uptake of stain is then quantified from the extracted lipids by spectrophotometry, which showed that adipogenesis in explant-derived ASC is comparable to that of collagenase-derived
  • FIG. 4c Induction of osteogenic differentiation resulted in matrix mineralization, and is confirmed by staining with Alizarin red S (Fig. 4d, e) that is then extracted and colorimetrically estimated (Fig. 4f).
  • Explant-derived ASC is found to be as competent as collagenase-derived ASC for osteogenic differentiation. Chondrocyte pellets obtained from both ASC is stained with alcian blue to visualize proteoglycan deposition (Fig. 4g, h). Gene expression analysis of the differentiated pellets confirmed equivalent expression of the chondrogenesis markers SOX9, collagen 1 and collagen X in both ASC preparations (Fig. 4i).
  • the method disclosed in present disclosure describes ASC is reliably isolated from explants of adipose tissue obtained by two different surgical procedures: resection and liposuction.
  • the expansion, phenotypic and functional properties of ASC obtained using the explant method of present disclosure are comparatively better, and the present disclosure is also far more economically viable than the conventional and widely used enzymatic techniques.
  • the proliferation potential of both explant and enzymatically derived ASC is nearly identical.
  • explant culture is advantageous for processing very small quantities of adipose tissue, as 5-10 xlO 5 enriched ASC could be derived from just 1 g of explant tissue. Further culturing yields an expansion of 10 4 fold over just 5 passages, which corresponded to 12.5 ⁇ 2 population doublings (Fig. 2).
  • ASC are known to express CD34 in the adipose tissue niche.
  • Freshly isolated SVF therefore contains CD34+ cells in the ASC fraction [Planat-Benard, et al (2004). Circulation, 109:656-663 and Mitchell, et al. (2006). Stem Cells, 24:376-385] and the expression of this marker has been shown to decline upon ex-vivo culture and expansion, accompanied by an increase in expression of the mesenchymal markers [Mitchell, et al. (2006). Stem Cells, 24:376-385].
  • Enzymatically derived SVF is also reported to contain undesired hematopoietic cells such as monocytes/macrophages and lymphocytes [Mitchell, et al. (2006).
  • ASC obtained by present explant technique shows significantly lower percentage of CD34+ and HLA- DR+ cells at P0 when compared to collagenase-derived ASC (Fig. 3) indicating an enriched MSC population. Further expansion of cells derived by both techniques showed nearly identical surface marker profiles validating the mesenchymal stem cell phenotype of explant-derived ASC.
  • ASC isolated by explant technique also possessed trilineage differentiation capacity comparable with collagenase-derived ASC (Fig.
  • the ASC obtained by explanting therefore fulfill the criteria for defining multipotent mesenchymal stromal cells as proposed by the International Society for Cellular Therapy namely, they are plastic adherent with spindle shaped fibroblastic morphology, express the cell surface antigens CD90, CD73 and CD105, and lack expression of hematopoietic markers. They also demonstrate trilineage differentiation into adipogenic, osteogenic and chondrogenic lineages.
  • Immune privilege and immunosuppressive capacity are two properties of tremendous therapeutic utility for MSC, which eliminates the requirement for histocompatibility matching. Allogeneic donor-derived MSC can thus be made readily available for acute clinical conditions, closing the lead time from the bench to the bedside.
  • ASC derived by explant culture is found to be negative for HLA-DR and immune co-stimulatory molecules CD80, CD86 and CD40 (Fig. 3), and they did not provoke stimulation of allogeneic lymphocytes confirming their low immunogenicity (Fig. 5). They were also capable of immunosuppression to a similar extent as collagenase-derived ASC.
  • the present explant culture method yields ASC, this technique also has several advantages over the conventional enzymatic technique from a clinical and regulatory perspective.
  • the tissue is not dissociated or processed using chemical or physical means, and thus places minimal stress on the cells.
  • the processing time is also very rapid, as the surgically removed tissue can be transferred to a physiologic environment (37°C, 5% C0 2 , isotonic culture medium containing relevant growth factors) with minimal time delay thus ensuring that the adipose tissue/MSC spends minimal time outside physiological environment (less than 30 min).
  • present disclosure of using explant method demonstrates that ASC can be efficiently isolated not only from resected fat tissue, but also from liposuctioned fat. As liposuction procedures are preferred over other invasive techniques of fat harvest, the combined methods of lipoaspiration and explanting has implications in reducing the cost associated with, and hugely simplifying, the ASC-isolation process.
  • Present disclosure shows explanting adipose tissue results in an enriched ASC population at P0. The migration of plastic adherent cells from the explant tissue served as the criterion for isolation, and resulted in significantly reduced hematopoietic cells and contaminants in ASC preparation using explants method of present disclosure.
  • explant-derived ASC are immune privileged and exhibit immunosuppressive activity critical for MSC-based therapy, particularly for allogeneic cell therapy. Therefore, presently disclosed explant culture method is an efficient, reproducible and economical technique of clinical utility, which may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem cells for tissue engineering and regenerative medicine.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé d'isolement et de culture de cellules souches/stromales mésenchymateuses issues de tissu adipeux (ASC) par un procédé de culture d'explant. Le tissu adipeux obtenu par un procédé chirurgical ou un autre procédé connu est traité pour isoler une population enrichie en MSC de façon plus économique. Les ASC multipotents ainsi obtenus par le procédé de culture d'explant sont capables d'auto-renouvellement, et sont immunosuppresseurs et hypo-immunogènes. Le procédé d'explant induit un stress minimal ou aucun stress sur les cellules souches contrairement aux procédés classiques.
PCT/IB2011/055252 2010-11-24 2011-11-23 Technique de culture d'explant pour l'isolement de cellules souches mésenchymateuses à partir de tissu adipeux WO2012070001A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN3544/CHE/2010 2010-11-24
IN3544CH2010 2010-11-24

Publications (1)

Publication Number Publication Date
WO2012070001A1 true WO2012070001A1 (fr) 2012-05-31

Family

ID=45464645

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2011/055252 WO2012070001A1 (fr) 2010-11-24 2011-11-23 Technique de culture d'explant pour l'isolement de cellules souches mésenchymateuses à partir de tissu adipeux

Country Status (1)

Country Link
WO (1) WO2012070001A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015081410A1 (fr) * 2013-12-05 2015-06-11 Fundação Universidade De Caxias Do Sul Procédé de culture de cellules souches mésenchymateuses adultes, trousse, composition et utilisations
CN104974984A (zh) * 2015-04-13 2015-10-14 深圳市默赛尔生物医学科技发展有限公司 一种脂肪组织来源的间充质干细胞的扩增培养方法
CN107083359A (zh) * 2017-06-14 2017-08-22 深圳市泰华细胞工程有限公司 干细胞培养基及干细胞分离方法
CN111763656A (zh) * 2020-07-24 2020-10-13 深圳市人和生物科技有限公司 脂肪来源间充质干细胞临床级提纯分离、培养扩增和冻存方法
WO2021185198A1 (fr) * 2020-03-16 2021-09-23 北京全式金生物技术有限公司 Milieu de culture de cellules souches mésenchymateuses exempt de sérum et de composants hétérologues et utilisation associée

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Retraction: Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.", JOURNAL OF MOLECULAR CELL BIOLOGY DEC 2011 LNKD- PUBMED:22128322, vol. 3, no. 6, December 2011 (2011-12-01), pages 382, XP055021470, ISSN: 1759-4685 *
"Technical Resources - Media Formulations 10569 - DMEM, high glucose, GlutaMAX(TM), pyruvate", 9 March 2012 (2012-03-09), XP055021491, Retrieved from the Internet <URL:http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation.46.html> [retrieved on 20120309] *
HE XIAOLI ET AL: "Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.", JOURNAL OF MOLECULAR CELL BIOLOGY AUG 2010 LNKD- PUBMED:20584786, vol. 2, no. 4, August 2010 (2010-08-01), pages 199 - 208, XP055021461, ISSN: 1759-4685 *
JING WEI ET AL: "Explant culture: an efficient method to isolate adipose-derived stromal cells for tissue engineering.", ARTIFICIAL ORGANS FEB 2011 LNKD- DOI:10.1111/J.1525-1594.2010.01054.X PUBMED:20946305, vol. 35, no. 2, 14 October 2010 (2010-10-14), pages 105 - 112, XP055021341, ISSN: 1525-1594 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015081410A1 (fr) * 2013-12-05 2015-06-11 Fundação Universidade De Caxias Do Sul Procédé de culture de cellules souches mésenchymateuses adultes, trousse, composition et utilisations
CN104974984A (zh) * 2015-04-13 2015-10-14 深圳市默赛尔生物医学科技发展有限公司 一种脂肪组织来源的间充质干细胞的扩增培养方法
CN107083359A (zh) * 2017-06-14 2017-08-22 深圳市泰华细胞工程有限公司 干细胞培养基及干细胞分离方法
WO2021185198A1 (fr) * 2020-03-16 2021-09-23 北京全式金生物技术有限公司 Milieu de culture de cellules souches mésenchymateuses exempt de sérum et de composants hétérologues et utilisation associée
CN111763656A (zh) * 2020-07-24 2020-10-13 深圳市人和生物科技有限公司 脂肪来源间充质干细胞临床级提纯分离、培养扩增和冻存方法

Similar Documents

Publication Publication Date Title
Trivedi et al. Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells
Priya et al. Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate
Ullah et al. Human mesenchymal stem cells-current trends and future prospective
JP5732011B2 (ja) 非骨軟骨性の間葉組織由来の多能性細胞の同定および単離
Iacono et al. Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse
Bunnell et al. Adipose-derived stem cells: isolation, expansion and differentiation
Bieback et al. Clinical protocols for the isolation and expansion of mesenchymal stromal cells
Gottipamula et al. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions
US20150175970A1 (en) Cells for therapy of the heart, method of obtaining a cell preparation, and cell preparation
CN109234229B (zh) 从胎盘血管分离间充质干细胞的方法和所用消化酶组合物
EP3019599B1 (fr) Méthode d&#39;isolement de fraction stroma-vasculaire
CN111454893B (zh) 一种无血清、无异源成分的间充质干细胞培养基及其应用
WO2012131618A1 (fr) Composition comprenant des cellules souches mésenchymateuses dérivées de la gelée de wharton et procédés associés
WO2012070001A1 (fr) Technique de culture d&#39;explant pour l&#39;isolement de cellules souches mésenchymateuses à partir de tissu adipeux
Pilgaard et al. Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells
JP6781752B2 (ja) 脂肪間質細胞を急速分離する組成物
CN109628388B (zh) 用消化酶组合物从胎盘血管分离间充质干细胞
WO2013102114A1 (fr) Procédé de production de cellules souches fraîches et congelées
Gottipamula et al. Mesenchymal Stromal Cells: Basics, Classification, and Clinical Applications.
US20080081370A1 (en) Directed differentiation of human embryonic stem cells into mesenchymal/stromal cells
EP3978599A1 (fr) Procédés permettant de favoriser la prolifération et la propagation de cellules souches
Van Den Berk et al. The tissue origin and culture history of mesenchymal stem cells affect their performance in osteoblastic differentiation
CN117305236A (zh) 脂肪间充质干细胞低氧气氛培养及其治疗卵巢早衰的用途
AU2011253985C1 (en) Identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue
No Optimization of Adipose-Derived Mesenchymal Stem Cells Harvest Protocol Toward Clinical Applications

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11805946

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11805946

Country of ref document: EP

Kind code of ref document: A1