WO2013149957A1 - Milieux de culture améliorés pour cellules embryonnaires - Google Patents

Milieux de culture améliorés pour cellules embryonnaires Download PDF

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WO2013149957A1
WO2013149957A1 PCT/EP2013/056779 EP2013056779W WO2013149957A1 WO 2013149957 A1 WO2013149957 A1 WO 2013149957A1 EP 2013056779 W EP2013056779 W EP 2013056779W WO 2013149957 A1 WO2013149957 A1 WO 2013149957A1
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culture medium
sgpl30
embryo
embryos
development
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Kjell WÅNGGREN
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Waanggren Kjell
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    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]

Definitions

  • the present invention relates generally to mammalian in vitro fertilization (IVF) and to culture media and processes useful for carrying out fertilization and embryo development.
  • IVF mammalian in vitro fertilization
  • the invention provides a culture medium comprising sgpl30 that supports the growth and development of embryos to the blastocyst stage and significantly increases the likelihood of successful pregnancy in IVF.
  • In vitro fertilization is a technique used to overcome various forms of male and female infertility.
  • the process involves fertilizing an oocyte with a sperm in vitro and subsequently transferring the developing embryo into the female body.
  • IVF Steptoe, P.C. and Edwards, R.G., Lancet 2(8085): 366 (1978)
  • the process faces continuing challenges of low implantation and pregnancy rates.
  • the reported success rate for IVF was only 8 to 10%. This low success rate was due, at least in part, to the poor quality of the embryo culture media.
  • Simple media are those, such as Earle's and human tubal fluid (HTF), which are balanced salt solutions with added carbohydrate energy sources such as pyruvate, lactate and glucose.
  • Complex media such as Ham's F-10, further include non-essential and essential amino acids as well as other additives, such as vitamins, antibiotics and serum or proteins.
  • IVF culture media are intended to support embryo development up to the 8-cell stage or beyond in a single medium
  • the trend has been to optimize separate culture media to support the developing embryo at different stages of development.
  • This has led to the widespread use of sequential culture medium in IVF.
  • a sequential culture media system may use one culture medium for the growth of an embryo from a one- cell zygote to an eight-cell embryo during the first 48 hours of development and another culture medium to grow the eight-cell embryo to the blastocyst stage.
  • the sequential culture media are designed to more closely mimic the female reproductive tract during in vivo embryo growth.
  • the media compositions typically differ with respect to components such as amino acids and sugars to improve optimization of the media to support the embryonic growth and development.
  • the benefits of sequential culture media also show how specialized embryo culture media should be to improve IVF outcomes.
  • the transmembrane glycoprotein 130 (gpl30) is a commonly used, signal transducing receptor unit, for the IL-6 type cytokines, LIF (Leukemia Inhibitor Factor), IL-6 (Interleukin 6), IL-11 (Interleukin 11), IL-27 (Interleukin 27), CNTF (ciliary neurothrophic factor), CT-1 (cardiothrophin-1), OSM (oncostatin-M) and CLC (cardiothropophine like cytokine) (Jones et al 2011). It is expressed in most organs and deletion of the gpl30 gene in mice results in embryonic lethality.
  • LIF-R is an example of the a-receptor subunit which has LIF as the ligand.
  • the molecular weight of the membrane bound form of gpl30 is ⁇ 150 KDa. However there is also soluble forms of gpl30 (sgpl30) with a molecular weight of ⁇ 100 KDa (Classen-Linke et al 2004).
  • the sgpl30 can be released by proteolysis from the cell membranes or can be produced through alternative mRNA splicing.
  • the concentration of sgpl30 in human serum is about 300 ng/ml (Muller-Neven et al 1998).
  • sgpl30 localized in the endometrium is released by proteolytic activity on the membrane bound form.
  • the effect of soluble sgpl30 in the endometrium is not known.
  • sgpl30 has been shown to bind to IL-6/sIL-6r complexes and block IL- 6 trans-signaling (Muller-Neven et al 1998).
  • the IL-6 trans-signaling pathway is a way for IL-6 to communicate with cells not presenting IL-6 receptors. This is possible through binding of the soluble IL-6/IL-6r complexes to membrane bound gpl30 which appears to be present on all cells.
  • the IL-6/IL-6r complexes also have the ability to bind to others of the gpl30 membrane bound heterodimers to bypass other ligands.
  • the IL-6r works agonistic to enhance IL-6 type cytokine signaling.
  • sgpl30 when sgpl30 is also present, it binds to the IL-6/IL-6r complexes and antagonize IL-6 signaling.
  • sgpl30 has been suggested for use in auto-immune disease and cancer therapy (Jones et al 2011).
  • Gpl30 and the some of the ligands for which it transduces signals are important in relation to embryonic development and/or implantation.
  • Gpl30 has been shown to be expressed on the endometrial and decidual tissue during mid to late secretory phase. The soluble form was detected in cultured endometrial and decidual epithelial cells originating from the proliferative phase. This release was regulated by steroid hormones. A combination of Estrogen and Progesterone induced highest level of release (Classen-Linke et al 2004). Furthermore it has been shown that sgpl30 is up-regulated during implantation and that unexplained infertility could be related to altered secretion (Sherwin et al 2002).
  • LIF and IL-11 have been implicated as critical for the implantation in mice and LIF has been shown to increase blastocyst development in various species like mice, cow and humans (Dunglison et al 1996).
  • LIF and LIFr have been shown to be expressed by bovine embryos. The expression was stronger in in vitro produced embryos and could be related to the decreased developmental potential of those embryos (Rizos et al 2002).
  • the mechanisms on which the IL-6 like cytokines acts on pre-implantation embryos and during implantation are complex and largely unknown and might partly be species specific.
  • gpl30 has been shown to be expressed on human oocytes but not on murine oocytes (Roushandheh et al 2010).
  • the present invention provides improved culture media for mammalian embryos and in particular for human embryos.
  • the invention further provides methods for culturing mammalian embryos and particularly human embryos in the culture media.
  • the culture media are characterized by comprising sgpl30 in the medium.
  • the culture medium includes water, inorganic salts, at least one energy source, and sgpl30 at a concentration of between 1 ng/ml to 1 mg/ml. Preferably between 10 ng/ml and 10 ⁇ g/ml, more preferably between 50 ng/ml and 1000 ng/ml and most preferably between 100 ng/ml and 500 ng/ml.
  • the medium further includes one or more amino acids.
  • the medium is deficient of exogenous LIF or contains less than 100 ng/ml of exogenous LIF.
  • the sgpl30 is substituted by a gp 130 antibody or by an IL-6 cytokine family antagonist.
  • the culture media may be tailored to support the growth of embryos from a first developmental stage to a second developmental stage.
  • a culture medium may be designed to support a mammalian embryo up to the 4-cell stage, up to the 8-cell stage or up to the blastocyst stage.
  • the various culture media encompassed by the present invention may include different components at varying concentrations.
  • the present invention provides IVF culture media compositions comprising sgpl30 in concentrations that support the development of gametes, zygotes and/or embryos in vitro prior to implantation.
  • the culture media and uses thereof described herein have significant noncommercial and non-industrial uses (e.g., research) in addition to (or as an alternative to) commercial and industrial uses.
  • This invention is not intended to support commercial uses of human embryos which are prohibited by law.
  • culture medium is used throughout the present specification to refer to aqueous media containing salts and carbohydrates prepared to a defined osmolarity and pH which is used for in vitro culture of mammalian embryos such as, gametes, zygotes, cleavage stage embryos, morulas and blastocysts.
  • Embryo culture media can be prepared in the laboratory by any practitioner skilled in the art and are also commercially available.
  • the composition of embryo transfer media is based on embryo culture media although transfer media generally comprises higher concentrations of hyaluronic acid i.e. 0.3 mg/ml to 5 mg/ml and preferably only recombinant proteins.
  • a transfer medium preferably comprises recombinant human albumin as the protein source. When they were originally used, embryo culture media often contained serum, and even contained a non-human serum such as fetal bovine serum. It has since been found that serums are not desirable in the embryo culture medium, particularly non-human serums.
  • the embryo culture medium of the present invention preferably does not include serum.
  • embryo is used to refer to cells in all stages of development from a fertilized egg up to the first 5 or 6 days or to the expanded or hatched blastocysts stages.
  • the invention is based, at least in part, on the inventor's discovery that culture media comprising sgpl30 substantially improves the success rate of IVF procedures compared to culture media that omit sgpl30.
  • the inventor also discovered that LIF, previously considered to be beneficial for blastocyst development, did not show any activity on human embryo development to the blastocyst stage and that the combination of LIF and sg l30 was detrimental to blastocyst development. The explanation for this negative synergy is difficult to comprehend. It could be an effect of too much LIF as LIF is also produced by the embryo.
  • the positive effect on blastocyst development is not primarily related to LIF activity, but to activity of any of the other IL-6 like cytokines.
  • the positive effect of sgpl30 could be associated to binding of the IL-6/IL-6r complexes antagonizing their effect.
  • the further addition of LIF could counteract that balance or sgpl30 might modify expression of gpl30 which causes an overreaction when exogenous LIF is also included in the system. If other members of the IL- 6 cytokine family are the primary target for sgpl30, then other ways to modify those targets could be useful to achieve the same effect.
  • Such alternative modifications could for example be achieved by specific antibodies or pharmaceuticals exhibiting similar inhibitory effects.
  • gpl30, IL-6, IL-11, IL-6r or IL-1 lr antibodies or a ligand antagonist could be antagonistic to the signal transducing pathways of gpl30 which might provide similar effects on in vitro embryo development as sgpl30.
  • the improvement provided by the present inventions is, when shown as an increase in blastocyst development from frozen human embryos, such that a 300 ng/ml concentration of sgpl30 results in 73% blastocyst development, compared to 37% in the control without sgpl30.
  • the blastocyst development was comparable to the control with 41%, but in the sgpl30 plus LIF comprising medium the blastocyst development was only 29%.
  • the percentage of the developed blastocysts that was considered to be high quality blastocysts was 38% in the sgpl30 comprising medium compared to 23% in the control.
  • the combined sgpl30 and LIF comprising medium produced less high quality embryos with only 17%.
  • Improvements provided by the present media may be measured by one or more of several parameters. These include increased survival rates or developmental rates for blastocysts up to the 4- to 6-day stage, blastocysts with a greater number of cells, blastocysts having larger inner cell masses and blastocyst having inner cell masses that make up a greater percent of the total and/or percentage of hatched blastocysts and increased proportion of high quality blastocysts. Improvements might also be shown through measuring of differential protein expression patterns.
  • the improvement in embryo development resulting from the use of the present culture media may be quantified by comparing embryos cultured in an improved medium comprising sgpl30 with embryos cultured in a control medium which differs from the improved medium only in that sgpl30 is absent from the control.
  • the present culture media may provide at least a 5%, at least a 10%, or even at least a 15% increase in one or more of the following: (1) percent compacting on day 3; (2) percent total blastocyst development on day 4; (3) percent total blastocyst development on day 5; and (4) percent blastocyst hatching on day 5.
  • the present media may provide blastocysts with a total inner cell number and/or an increase in the percent of the total mass due to the inner cell masses which are at least 10% higher, or even at least 15% higher, than those of blastocysts cultured in the control medium.
  • the culture media of the present invention may be optimized to support embryo development at various stages.
  • the media may be optimized to culture an embryo up to the 4-cell stage, up to the 8-cell stage, from the 4-cell stage to the 8-cell stage, up to the blastocyst stage, or from the 4- or 8- cell stage to the blastocyst stage.
  • the nature and concentration of the essential and non-essential ingredients of the culture media may vary, however, in each culture medium that includes sgpl30.
  • the sgpl30 may be present in one of the sequential media and absent in others.
  • the sgpl30 is supplemented to a non-sequential type medium, in which case the same medium is used for more than one developmental stage.
  • the culture media is suitable for human use.
  • the media may comprise one or more
  • the macromolecules may be present in combination with one or more proteins or may be used as an alternative to the proteins. Suitable macromolecules include, but are not limited to, extracellular matrix components such as heparin sulfate, fibronectin, laminin, collagen, elastin, hyaluronic acid and vitronectin or carbohydrate polymers such as dextran, or cellulose or starch derivatives such as hydroxy ethyl starch, or synthetic polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).
  • the macromolecules can be added to the media at a concentration of 0.01 mg/ml to 20 mg/ml, preferably 0.1 mg/ml to 10 mg/ml.
  • a typical culture medium for culturing embryos can include water, one or more inorganic salts, one or more energy sources, and one or more amino acids (including nonessential and, optionally, essential amino acids), as core ingredients.
  • Typical energy sources in an embryo culture medium include carbohydrate energy sources, such as pyruvate, lactate and glucose.
  • Suitable inorganic salts include, CaCl 2 ⁇ 2H 2 0, CuS0 4 ⁇ 5H 2 0, FeS0 4 ⁇ 7H 2 0, KC1, MgS0 4 , NaCl, NaHC0 3 , Na 2 HP0 4 , ZnS0 4 ⁇ 7H 2 0, and KH 2 P0 4 .
  • Non-essential amino acids include alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tryosine.
  • Essential amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
  • the culture media can include other additives.
  • vitamins such as biotin, pantothenate, folic acid, niacinamide, pyridoxine, riboflavin, and thiamine
  • growth factors such as progesterone and/or estrogen
  • antibiotics such as penicillin and streptomycin.
  • the antibiotic would be penicillin G or streptomycin sulfate or a combination thereof.
  • Albumin e.g., human serum albumin or recombinant human albumin
  • polyvinylpyrolidone e.g., polyvinylpyrolidone, hyaluronan, chelators, such as EDTA
  • buffers such as HEPES buffer or MOPS buffer
  • miscellaneous ingredients that also may be included in the culture media include, but are not limited to, one or more of choline chloride, hypoxanthine, inositol, thymidine, cyanocobalamine, cysteamine, phenol red, lipoic acid and glutathione.
  • the concentration of the core ingredients and other additives in a culture medium may vary depending upon the stage of embryo development for which the medium is optimized.
  • Typical concentrations for the inorganic salts in the culture media may be about 100 mM to about 150 mM or about 110 mM to about 140 mM.
  • Typical concentrations for the energy sources in the media may be about 5 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 15 mM.
  • Typical concentrations for the total amount of amino acids in the culture media may be about 0.1 mM to about 15 mM or about 0.5 mM to about 12 mM, or about 0.5 mM to about 6 mM.
  • the present culture media may be formulated by adding sgpl30 to existing embryo culture media.
  • the sgpl30 is preferably human recombinant sgpl30, however other sgpl30 preparations could be used.
  • sgpl30 can be added to commercially-available simple culture media, such as human tubal fluid (HTF), Whittingham's T6 medium and Earle's balanced salt solution (EBSS) (available from Irvine Scientific) that have been supplemented with pyruvate, glucose and lactate as energy sources and protein or another macromolecule providing similar effect as the protein.
  • HVF human tubal fluid
  • EBSS Earle's balanced salt solution
  • These simple media have generally been employed in clinical IVF settings for oocyte insemination and cultivation of embryos until transfer on day 2 or 3.
  • the sgpl30 may also be added to more complex embryo culture media designed to support early embryo growth.
  • Such media include GlTM culture medium available from Vitrolife. This medium is designed to support the development of cleavage stage embryos to around the 8-cell stage.
  • the medium contains carbohydrates, amino acids, and chelators to support the early embryo.
  • the complete formulation for the GlTM medium is provided in Table 1.
  • sgpl30 may be added to commercially available IVF culture medium capable of supporting embryos to beyond day 3 (8-cell stage). These culture media have been designed to carry embryos to the blastocyst stage prior to implantation. In accordance with the present invention, sgpl30 may be added to these media to enhance the blastocyst development.
  • One example includes a-modified essential medium ( MEM), described in Desai et ah, Human Reprod 12: 328-335 (1997).
  • a second example includes HECM-6 medium plus pantothenate. McKiernan SH and Bavister BD, Human Reprod 15: 157-164 (2000).
  • a third example includes CCM medium available from Vitrolife.
  • G2TM is a medium that is designed to support the development of the embryo from around the 8-cell stage (day 3) to the blastocyst stage.
  • the medium (Table 2) contains carbohydrates, amino acids, and vitamins to support the later stage embryo.
  • 8-cell stage embryos are transferred from an embryo culture medium optimized to support early stage growth (i.e., up to the 8-cell stage) supplemented with sgpl30 to an embryo culture medium optimized to support later stage growth (i.e., up to the blastocyst stage) sg l30 may or may not be added to the latter media.
  • the sg l30 can be added to non-sequential culture medium using the same medium for one or more stages of development.
  • An example of such a non-sequential culture medium is Global® available from LifeGlobal.
  • the present culture media also may be formulated by adding sgpl30 to existing gamete (oocyte or sperm) culture or freezing media. Tables 3-5 below provide gamete culture media in which sgpl30 may be included to provide for improved cellular
  • Down-regulation for ovarian hyper stimulation was achieved by using a gonadotropin- releasing hormone (GnRH) agonist (Suprecur Hoechst, Frankfurt, Germany, Synarela, Syntex Nordica AB, Sodertalje, Sweden), starting on cycle day 21.
  • GnRH gonadotropin- releasing hormone
  • ovarian stimulation was induced by using either recombinant FSH, (Gonal-F, Serono Laboratories, Aubonne, Switzerland, or Puregon, Organon, Oss, the Netherlands) or urine- derived Menopur® (Saint-Prex, Switzerland). The starting dose was dependent on the woman's age and/or previous response to ovarian stimulation.
  • the ovarian response was monitored by means of serum estradiol assays and vaginal ultrasonographic scans of follicles. Recombinant FSH or Menopur® was administered until the leading follicle had a diameter of at least 17 mm. Maturation of the oocyte was triggered by s.c. injection of hCG (Pregnyl, Organon, Oss, the Netherlands or Ovitrelle, Serono Laboratories, Aubonne, Switzerland). Oocytes were retrieved by transvaginal needle aspiration under ultrasonographic guidance 35-36 hours after hCG administration.
  • hCG Pregnyl, Organon, Oss, the Netherlands or Ovitrelle, Serono Laboratories, Aubonne, Switzerland.
  • embryos Prior to cryopreservation, embryos were cultured in microdroplets (Gl v5, Vitrolife AB, Frolunda, Sweden) overlaid with mineral oil (OVOIL) at 37C and 6% C02 in a humidified incubator.
  • the embryos were cryopreserved on day 2-3 (48-72 hrs after ovum pick up) according to a controlled rate freezing protocol involving the use of 1, 2 propanediol and sucrose solution in phosphate-buffered saline (PBS) as cryoprotectants (Vitrolife AB, Frolunda, Sweden). All embryos used in the study were frozen for at least 5 years.
  • PBS phosphate-buffered saline
  • the thawing protocol was performed using a commercial kit according to the instructions from the manufacturer (Thawing Kit, Cook Medical, Brisbane, Australia). The embryos were then transferred into equilibrated culture medium CCM (Vitrolife AB, V. Frolunda, Sweden). Thawed embryos were then carefully assessed for blastomere survival and then randomly allocated, with regard to embryo morphology/survival rate, into four different treatment regimes and cultured up to blastocyst stage.
  • CCM equilibrated culture medium
  • the four groups were control medium alone or supplemented with LIF,Human, Recombinant, 100 ng/ml (SIGMA, Saint Louis, Missouri, USA) and/or sgpl30, Human, Recombinant, 300 ng/ml (SIGMA, Saint Louis, Missouri, USA).
  • the embryos were cultured as single embryos in a 20 droplet and assessed daily for developmental stage. Culture was performed at 37°C in a humidified incubator (1st part) or with continuous monitoring of embryo development using EmbryoScope® time lapse equipment (Unisense FertiliTech, Aarhus, Denmark) (2nd part).
  • Mc Nemar's Test was used for morphological evaluation using IBM SPSS Statistics 20. Results For the present study, a total of 164 embryos survived after thawing and were cultured for four days which corresponds to day 6 after insemination. Of these, 73 embryos developed into blastocysts (44 %) and 38 (24 %) were of good quality, i.e. fully expanded or hatching with good quality inner cell mass and trophectoderm (Table I). Culture of embryos in media supplemented with sgpl30 resulted in a significant higher number of embryos developed into blastocysts (73 %) compared to controls (37 %), LIF (41 %) or LIF+sgpl30 (29 %) respectively (Table I). Culture with LIF was not beneficial for embryo development, there was no difference compared to control (Table I).

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Abstract

La présente invention concerne une composition et un procédé pour la fertilisation in vitro qui utilise des milieux de culture comprenant sgp 130. Des milieux de culture d'embryon qui comprennent sgp 130 sont capables de produire un taux de développement de blastocystes améliorés ainsi que d'augmenter la production de blastocystes de haute qualité.
PCT/EP2013/056779 2012-04-05 2013-03-28 Milieux de culture améliorés pour cellules embryonnaires WO2013149957A1 (fr)

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DESAI ET AL., HUMAN REPROD, vol. 12, 1997, pages 328 - 335
JEONG YOON ET AL: "Application of two different synthetic sequential media for the human IVF-ET program: a prospective, randomized, and comparative study", CLINICAL AND EXPERIMENTAL REPRODUCTIVE MEDICINE, vol. 38, no. 4, 1 January 2011 (2011-01-01), pages 186, XP055062414, ISSN: 2233-8233, DOI: 10.5653/cerm.2011.38.4.186 *
MCKIERNAN SH; BAVISTER BD, HUMAN REPROD, vol. 15, 2000, pages 157 - 164
MICHAL AMIT ET AL: "Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells", STEM CELL REVIEWS AND REPORTS, vol. 6, no. 2, 30 April 2010 (2010-04-30), pages 248 - 259, XP055015267, ISSN: 1550-8943, DOI: 10.1007/s12015-010-9149-y *
MIHO SUZUKI ET AL: "Anti-inflammatory mechanism of tocilizumab, a humanized anti-IL-6R antibody: effect on the expression of chemokine and adhesion molecule", RHEUMATOLOGY INTERNATIONAL ; CLINICAL AND EXPERIMENTAL INVESTIGATIONS, SPRINGER, BERLIN, DE, vol. 30, no. 3, 23 May 2009 (2009-05-23), pages 309 - 315, XP019779564, ISSN: 1437-160X *
SHUHONG ZHANG ET AL: "The novel histone deacetylase inhibitor, AR-42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells", INTERNATIONAL JOURNAL OF CANCER, vol. 129, no. 1, 1 July 2011 (2011-07-01), pages 204 - 213, XP055062409, ISSN: 0020-7136, DOI: 10.1002/ijc.25660 *
STEPTOE, P.C.; EDWARDS, R.G., LANCET, vol. 2, no. 8085, 1978, pages 366

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