US20110171731A1 - Compositions containing platelet contents - Google Patents

Compositions containing platelet contents Download PDF

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US20110171731A1
US20110171731A1 US13/119,350 US200913119350A US2011171731A1 US 20110171731 A1 US20110171731 A1 US 20110171731A1 US 200913119350 A US200913119350 A US 200913119350A US 2011171731 A1 US2011171731 A1 US 2011171731A1
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platelet
cells
medium
percent
plasma
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Allan B. Dietz
Michael P. Gustafson
Greg W. Butler
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Mayo Foundation for Medical Education and Research
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • 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
    • 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
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • This document relates to methods and materials involved in making and using growth factors, chemokines, and molecules responsible for growing, differentiating, or maintaining undifferentiated cells from normal human platelets (e.g., platelet lysates).
  • this document relates to methods and materials for manufacturing from platelets or platelet preparations (e.g., platelet apheresis preparations) those factors used to grow stem cells (e.g., adult stem cells) rapidly, to maintain them in an undifferentiated form, as an additive to media to differentiate stem cells (e.g., adult stem cells) in combination with other factors, to grow primary cell cultures (e.g., tumor cells and tumor cell lines), and to grow tumor cells with stem cell properties.
  • This document also relates to methods and materials that can be used to identify, isolate, enrich, or optimize combinations of effective growth factors using platelets as a source material.
  • the document relates to platelet plasma culture supplements and compositions containing platelet contents.
  • primary tumor cells can be cultured to obtain an antigen source that can be used in anti-tumor vaccines.
  • Two general methods are used to culture primary brain tumor cells. Traditionally, cells are grown in minimal media supplemented with fetal bovine serum. Cells grown in this way can exhibit limited applicability as an effective antigen source because they often exhibit characteristics of differentiated glial cell subtypes with a reduced ability to recapitulate the original tumor in vivo.
  • cells can be grown in an enriched media supplemented with growth factors, EGF, and FGF. Cells grown in this environment can form neurospheres and can contain populations of tumor stem cells, which more closely recapitulate the phenotypic characteristics of the primary tumor.
  • these cells can have reduced proliferative capacities, they are typically less differentiated, more tumorigenic, and more antigenic than cells grown in fetal bovine serum (Lee et al., Cancer Cell., 9:391-403 (2006)). These approaches do not appear to generate primary tumor cultures with high efficiencies and do not appear to allow for the growth of cultures fast enough for many applications. While these methods can be used to generate cell cultures from malignant gliomas, these protocols typically include materials and methods not suitable for clinical use.
  • This document provides methods and materials relating to the collection and use of molecules and growth factors found in platelets (e.g., platelet lysates or “PL”) or media containing platelets (e.g., a supernatant or filtrate from a platelet preparation such as a platelet apheresis preparation).
  • platelets e.g., platelet lysates or “PL”
  • media containing platelets e.g., a supernatant or filtrate from a platelet preparation such as a platelet apheresis preparation
  • compositions containing platelet contents e.g., platelet lysates and/or supernatants or filtrates from platelet-containing media.
  • the compositions containing platelet contents provided herein can be used to promote enhanced growth of cells (e.g., normal adult progenitor cells, stem cells, and precursor cells).
  • compositions containing platelet contents provided herein also can be used to enhance differentiation of progenitor cells, stem cells, and precursor cells into functional subtypes efficiently.
  • the compositions containing platelet contents provided herein can be used to promote enhanced growth of undifferentiated stem cells such as tumor stem-like cells and adult mesenchymal stromal cells or to maintain cells as a mixture of stem cells and differentiated cells such as that found in primary tumor cultures and tumor cell lines.
  • the compositions containing platelet contents provided herein can be used to generate primary cell cultures (e.g., tumor cell lines) efficiently.
  • the compositions containing platelet contents provided herein can be used to obtain superior growth kinetics when culturing cells (e.g., progenitor cells and tumor stem-like cells) as compared to cultures using supplementation from human serum, fetal bovine serum, or serum-free media with recombinant growth factor supplementation.
  • the compositions containing platelet contents provided herein also can be manufactured in a manner that is acceptable for clinical use (e.g., meets current good manufacturing practice regulations).
  • platelet plasma culture supplement refers to a composition that contains (a) plasma (e.g., human plasma) and (b) platelet content that is in the form of a platelet lysate that contained greater than 3 ⁇ 10 8 platelets per mL (e.g., greater than 4 ⁇ 10 8 platelets per mL, greater than 5 ⁇ 10 8 platelets per mL, greater than 6 ⁇ 10 8 platelets per mL, greater than 7 ⁇ 10 8 platelets per mL, greater than 8 ⁇ 10 8 platelets per mL, greater than 9 ⁇ 10 8 platelets per mL, or greater than 1 ⁇ 10 9 platelets per mL) before lysis or a medium that contained greater than 3 ⁇ 10 8 platelets per mL (e.g., greater than 4 ⁇ 10 8 platelets per mL, greater than 5 ⁇ 10 8 platelets per mL, greater than 6 ⁇ 10 8 platelets per mL
  • Such a medium can be a supernatant or filtrate obtained from a platelet preparation such as a platelet apheresis preparation or a large scale platelet isolation preparation.
  • a platelet preparation such as a platelet apheresis preparation or a large scale platelet isolation preparation.
  • supernatants or filtrates can be obtained from outdated platelet preparations (e.g., outdated platelet apheresis preparations).
  • Outdated platelet preparations can be platelet preparations (e.g., platelet apheresis preparations) that have been obtained from a live human and stored between 20° C. and 24° C. for more than four days (e.g., more than five, more than six, seven, eight, nine, ten, 11, 12, 15, or more days).
  • a preparation containing platelets at the above concentrations can be used.
  • 30 mL e.g., two 15-mL tubes
  • 50 mL, 100 mL, 500 mL, or an entire unit of platelets can be used to obtain a platelet lysate or a medium (e.g., plasma) that contained greater than 3 ⁇ 10 8 platelets per mL.
  • a platelet plasma culture supplement provided herein can have the ability to establish glioma cultures.
  • minimal essential medium having 5 percent of a platelet plasma culture supplement provided herein can be capable of establishing, from newly diagnosed human glioblastoma multiforme biopsy tissue, human glioma cultures having greater than 3 ⁇ 10 7 cells within 60 days at 37° C. (in a humidified incubator with 5% CO 2 ) with a success rate that is greater than 30 percent (e.g., greater than 35, 40, 45, 50, 55, 60, 65, or more percent).
  • This success rate is based on the number of different newly diagnosed human glioblastoma multiforme biopsy tissue samples (i.e., samples for different humans) that can be used to establish a glioma culture, as opposed to the number of attempts using a single human's glioblastoma multiforme biopsy tissue sample multiple times.
  • the methods and materials provided herein can be used to grow adult stem cells rapidly, to differentiate stem cells (e.g., adult stem cells), to grow primary cell cultures (e.g., tumor cell lines), to grow tumor stem-like cells, and to identify effective growth factors.
  • stem cells e.g., adult stem cells
  • primary cell cultures e.g., tumor cell lines
  • tumor stem-like cells e.g., tumor stem-like cells
  • the methods and materials provided herein can allow clinicians or medical personnel to develop patient-specific autologous cancer vaccines, while following FDA guidelines.
  • Tumor cells such as malignant glioma cells grown using the methods and materials provided herein can maintain many aspects of neural tumor stem cell phenotypes and can be enriched in tumor-specific antigens desired for recognition of host immune responses.
  • the growth kinetics of cells grown using the methods and materials provided herein can allow clinicians to manufacture sufficient cellular material for multiple vaccinations in a short time frame dictated by current standard therapeutic regimens.
  • the methods and materials provided herein can provide additional options for the expansion and use of patient specific tumor material for cell therapy.
  • stem cells both adult and fetal
  • stem cells can be used in regenerative medicine.
  • speed of cell growth, fidelity of culture genetics, and clinical applicability can be involved in the success of the cell based therapies.
  • a platelet lysate composition comprising, or consisting essentially of, a filtrate from a lysed platelet preparation passed through a 0.45 ⁇ m or smaller filter.
  • the lysed platelet preparation can be a lysed apheresis platelet preparation.
  • the filter can be a 0.45 ⁇ m filter.
  • the filter can be a 0.2 ⁇ m filter.
  • the filtrate can be from the lysed platelet preparation being passed through a 0.45 ⁇ m filter and a 0.2 ⁇ m filter.
  • the lysed platelet preparation can comprise supernatant from centrifugation of lysed platelets.
  • the lysed platelets can be platelets lysed via a freeze/thaw cycle.
  • the lysed platelets can be platelets lysed via at least two freeze/thaw cycles.
  • the centrifugation can comprise a force between 2000 ⁇ g and 4000 ⁇ g for between 15 and 45 minutes.
  • the centrifugation can comprise a force of about 3000 ⁇ g for about 30 minutes.
  • the platelet lysate composition can comprise greater than 200 pg of VEGF polypeptide per mL.
  • Culturing 1.4 ⁇ 10 6 mesenchymal stem cells with media containing about five percent of the platelet lysate composition can result in greater than 1.4 ⁇ 10 7 cells after three days.
  • the mesenchymal stem cells can be adipose derived cells.
  • this document features a platelet lysate composition produced by filtering a lysed platelet preparation through a 0.45 ⁇ m or smaller filter.
  • the lysed platelet preparation can be a lysed apheresis platelet preparation.
  • the filter can be a 0.45 ⁇ m filter.
  • the filter can be a 0.2 ⁇ m filter.
  • the platelet lysate composition can be produced by filtering the lysed platelet preparation through a 0.45 ⁇ m filter and a 0.2 ⁇ m filter.
  • the lysed platelet preparation can comprise supernatant from centrifugation of lysed platelets.
  • the lysed platelets can be platelets lysed via a freeze/thaw cycle.
  • the lysed platelets can be platelets lysed via at least two freeze/thaw cycles.
  • the centrifugation can comprise a force between 2000 ⁇ g and 4000 ⁇ g for between 15 and 45 minutes.
  • the centrifugation can comprise a force of about 3000 ⁇ g for about 30 minutes.
  • this document features a method for making a platelet lysate composition.
  • the method comprises, or consists essentially of, filtering a lysed platelet preparation through a 0.45 ⁇ m or smaller filter.
  • the lysed platelet preparation can be a lysed apheresis platelet preparation.
  • the filter can be a 0.45 ⁇ m filter.
  • the filter can be a 0.2 ⁇ m filter.
  • the method can comprise filtering the lysed platelet preparation through a 0.45 ⁇ m filter and a 0.2 ⁇ m filter.
  • the lysed platelet preparation can comprise supernatant from centrifugation of lysed platelets.
  • the lysed platelets can be platelets lysed via a freeze/thaw cycle.
  • the lysed platelets can be platelets lysed via at least two freeze/thaw cycles.
  • the centrifugation can comprise a force between 2000 ⁇ g and 4000 ⁇ g for between 15 and 45 minutes.
  • the centrifugation can comprise a force of about 3000 ⁇ g for about 30 minutes.
  • this document features a method for making a platelet lysate composition.
  • the method comprises, or consists essentially of: (a) lysing platelets via one or more freeze/thaw cycles to obtain lysed platelets, (b) centrifuging the lysed platelets to obtain a supernatant, and (c) filtering the supernatant through a 0.45 ⁇ m or smaller filter to obtain a filtrate, wherein the filtrate is the platelet lysate composition.
  • the lysing step can comprise at least two freeze/thaw cycles.
  • the centrifuging step can comprise using a force between 2000 ⁇ g and 4000 ⁇ g for between 15 and 45 minutes.
  • the centrifuging step can comprise a force of about 3000 ⁇ g for about 30 minutes.
  • the filter can be a 0.45 ⁇ m filter.
  • the filter can be a 0.2 ⁇ m filter.
  • the filtering step can comprise filtering the supernatant through a 0.45 ⁇ m filter and a 0.2 ⁇ m filter.
  • the platelets can be apheresis platelets.
  • this document features a method for expanding a cell population comprising, or consisting essentially of, culturing a first population of cells in the presence of medium comprising a platelet lysate composition under conditions wherein the first population of cells is expanded to a second population of cells having more cells than the first population, wherein the platelet lysate composition comprises a filtrate from a lysed platelet preparation passed through a 0.45 ⁇ m or smaller filter.
  • the cells can be adult stem cells, primary tumor cells, or tumor stem cells.
  • this document features a method for differentiating stem cells.
  • the method comprises, or consists essentially of, culturing the stem cells in the presence of differentiating medium comprising a platelet lysate composition under conditions wherein the stem cells differentiate, wherein the platelet lysate composition comprises a filtrate from a lysed platelet preparation passed through a 0.45 ⁇ m or smaller filter.
  • this document features a method for healing a wound.
  • the method comprises, or consists essentially of, contacting the wound with a platelet lysate composition, wherein the platelet lysate composition comprises a filtrate from a lysed platelet preparation passed through a 0.45 ⁇ m or smaller filter.
  • the wound can be a cut, fistula, or diabetic ulcer.
  • the contacting step can comprise using a stitch or glue comprising the platelet lysate composition to contact the wound.
  • the contacting step can comprise spraying the platelet lysate composition onto the wound.
  • this document features a composition containing plasma and platelet contents from a platelet preparation passed through a 0.45 ⁇ m or smaller filter.
  • the plasma can be human plasma.
  • the platelet contents can be human platelet contents.
  • this document features a composition comprising medium (e.g., a minimal essential medium) and a platelet plasma culture supplement.
  • the platelet plasma culture supplement can be a platelet lysate comprising plasma.
  • the platelet plasma culture supplement can be a filtrate obtained from filtering a platelet apheresis preparation comprising plasma.
  • the platelet apheresis preparation can be a platelet apheresis preparation collected from a human over three days earlier.
  • the platelet apheresis preparation can be a platelet apheresis preparation collected from a human over five days earlier.
  • the platelet apheresis preparation can be a platelet apheresis preparation collected from a human over six days earlier.
  • the platelet plasma culture supplement can be a supernatant obtained from spinning a platelet apheresis preparation comprising plasma.
  • the platelet apheresis preparation can be a platelet apheresis preparation collected from a human over ten days earlier.
  • this document features a method for obtaining a platelet plasma culture supplement comprising removing platelets or platelet debris from a platelet apheresis preparation such that at least some of the plasma within the platelet apheresis preparation is retained, thereby obtaining the platelet plasma culture supplement, wherein the platelet plasma culture supplement comprises the ability to induce differentiation of 1 ⁇ 10 6 adult stem cells into at least 1 ⁇ 10 3 progenitors of cardiomyocytes, adipocytes, ostoeblasts, chondrocytes, myocytes, or nerve cells within 60 days when added to medium at a concentration of 5 percent as a base media with supplementation directed at the desired cell type.
  • this document features an isolated cell culture comprising MCGBM103 cells cultured in medium comprising a platelet plasma culture supplement.
  • this document features an isolated cell culture comprising MCGBM106 cells cultured in medium comprising a platelet plasma culture supplement.
  • this document features an isolated cell culture comprising cells cultured in medium comprising a platelet plasma culture supplement, wherein the cells grow in the medium at a rate that is faster than the rate of control cells cultured in control medium comprising 10 percent fetal bovine serum.
  • the cells and the control cells can be primary tumor cells.
  • the cells and the control cells can be human primary glioma cells.
  • the cells and the control cells can be stem cells.
  • the cells and the control cells can be fibroblast cells.
  • the medium and the control medium can be neurobasal medium or DMEM/F12 medium. 1 ⁇ 10 5 of the cells can grow in the medium to more than 1 ⁇ 10 6 of the cells faster than 1 ⁇ 10 5 of the control cells cultured in the control medium grow to 1 ⁇ 10 6 of the control cells.
  • 1 ⁇ 10 5 of the cells can grow in the medium to more than 3 ⁇ 10 7 of the cells faster than 1 ⁇ 10 5 of the control cells cultured in the control medium grow to 3 ⁇ 10 7 of the control cells.
  • the cells can be primary glioma cells, and 5 ⁇ 10 5 of the cells can grow in the medium to more than 1 ⁇ 10 7 of the cells within 30 days.
  • the cells can be primary glioma cells, and 5 ⁇ 10 5 of the cells can grow in the medium to more than 1 ⁇ 10 7 of the cells within 60 days.
  • the cells can be stem cells, and 1 ⁇ 10 5 of the cells can grow in the medium to more than 1 ⁇ 10 7 of the cells within 14 days.
  • the cells can be stem cells, and 1 ⁇ 10 5 of the cells can grow in the medium to more than 1 ⁇ 10 7 of the cells within 20 days.
  • the stem cells can be mesenchymal stem cells.
  • the stem cells can be adipose-derived, mesenchymal stem cells.
  • the cells can grow in the medium at a rate that is 1.5 times faster than the rate of the control cells cultured in the control medium.
  • this document features an isolated cell culture comprising tumor cells cultured in medium comprising a platelet plasma culture supplement, wherein the cells grow in the medium in a manner that is more genetically stable than when control cells are cultured in control medium comprising 10 percent fetal bovine serum.
  • the tumor cells and the control cells can be primary tumor cells.
  • the tumor cells and the control cells can be human primary glioma cells.
  • the medium and the control medium can be neurobasal medium or DMEM/F12 medium.
  • the tumor cells grown in the medium for four doublings can exhibit less than a five percent change in karyotype as compared to the tumor cells grown in the medium for one doubling.
  • the tumor cells grown in the medium for four doublings can exhibit less than a three percent change in karyotype as compared to the tumor cells grown in the medium for one doubling.
  • the tumor cells grown in the medium for ten doublings can exhibit less than a five percent change in karyotype as compared to the tumor cells grown in the medium for one doubling.
  • this document features an isolated cell culture comprising stem cells cultured in medium comprising a platelet plasma culture supplement, wherein the stem cells grow in the medium in a manner that is more genetically stable than when control stem cells are cultured in control medium comprising 10 percent fetal bovine serum.
  • the stem cells and the control stem cells can be mesenchymal stem cells.
  • the stem cells and the control stem cells can be adipose-derived, mesenchymal stem cells.
  • the medium and the control medium can be neurobasal medium or DMEM/F12 medium.
  • the stem cells grown in the medium for four doublings can exhibit less than a five percent change in karyotype as compared to the stem cells grown in the medium for one doubling.
  • the stem cells grown in the medium for four doublings can exhibit less than a three percent change in karyotype as compared to the stem cells grown in the medium for one doubling.
  • the stem cells grown in the medium for ten doublings can exhibit less than a five percent change in karyotype as compared to the stem cells grown in the medium for one doubling.
  • this document features a method for obtaining a platelet plasma culture supplement.
  • the method comprises, or consists essentially of, removing platelets or platelet debris from a platelet apheresis preparation such that at least some of the plasma within the platelet apheresis preparation is retained, thereby obtaining the platelet plasma culture supplement, wherein the platelet plasma culture supplement comprises the ability to induce proliferation of less than 1 ⁇ 10 6 adipose-derived, mesenchymal stem cells at passage five into at least 1 ⁇ 10 8 cells within 20 days when added to minimal essential medium at a concentration of 5 percent.
  • the platelet plasma culture supplement can comprise the ability to induce proliferation of less than 1 ⁇ 10 6 adipose-derived, mesenchymal stem cells at passage five into at least 1 ⁇ 10 8 cells within 15 days when added to minimal essential medium at a concentration of 5 percent.
  • this document features a method for obtaining a platelet plasma culture supplement.
  • the method comprises, or consists essentially of, removing platelets or platelet debris from a platelet apheresis preparation such that at least some of the plasma within the platelet apheresis preparation is retained, thereby obtaining the platelet plasma culture supplement, wherein minimal essential medium having 5 percent of the platelet plasma culture supplement is capable of establishing, from newly diagnosed human glioblastoma multiforme biopsy tissue, human glioma cultures having greater than 3 ⁇ 10 7 cells within 60 days at 37° C. with a success rate that is greater than 30 percent. The success rate can be greater than 40 percent.
  • FIG. 1 Autologous primary neurospheres exhibited enhanced growth kinetics and characteristics of tumor stem cells.
  • A Morphology of a GBM tumor split and grown independently in Neural Stem Cell (NSC) media (neurospheres), HCTL#3 (mostly adherent cells in this particular cell culture; grown using 5% platelet lysate), and DMEM+10% FBS (monolayer forming adherent cells). Light phase photomicrographs, 10 ⁇ .
  • B Growth kinetics of GBM tumor grown using the three different methods.
  • C A table listing staining results for neural stem cell and other cell surface markers. Cells were grown on coverslips, and nestin and SOX2 expression was analyzed by indirect immunofluorescence.
  • FIG. 2 Identification and monitoring of tumor associated antigens in autologous primary brain tumor cultures. Gene chip analysis was used to determine relative antigen expression in primary tumors (black bars), tumor stem cell cultures obtained using the Fine protocol (Lee et al., Cancer Cell., 9:391-403 (2006)) (open bars; also known as NSC medium, Neural basal medium, neural stem cell medium), and brain tumor cultures using HCTL#1 (filled bars; grown using platelet lysate, and base media was DMEM/F12).
  • FIG. 3 is a graph plotting the total number of viable GBM cells after one, two, three, four, or five days of being cultured with the media containing the indicated components.
  • FIG. 4 is a graph plotting the total number of viable GBM cells after five days of being cultured with the indicated media.
  • FIG. 5 is a graph plotting the total number of MSCs after three, four, or five days of being cultured with the media containing the indicated components.
  • FIG. 6 is a graph plotting the number of GBM cells after three, six, and nine days of being cultured with the Neurobasal Media (Invitrogen, Grand Island, N.Y.) containing the indicated components.
  • FIG. 7 is a graph plotting the percent of CD133, CD105, and CD90 positive GBM cells after being cultured with the Neural Basal Media containing the indicated components.
  • FIG. 8 is a graph plotting the amount of VEGF (pg/mL) present in the indicated preparations.
  • MM minimally manipulated apheresis platelets from expired donations that were not frozen (3 bags were combined).
  • PPP Platelet poor plasma, which is a fraction of minimally manipulated apheresis platelets that were spun down at 3000 g for five minutes and then spun at 10,000 g for one minute.
  • FT freeze/thaw fraction, which is minimally manipulated apheresis platelets that were subjected to two freeze/thaw cycles.
  • PL platelet lysate prepared as described in Example 2. MM, PPP, FT, and PL were obtained from the same starting material and were stored for over two months at 4° C.
  • FIG. 9 demonstrates that the levels of growth factors (VEGF and PDGF) are consistent across different lots of manufactured platelet lysates.
  • the levels of these growth factors are significantly higher in the final product preparations versus other growth factor supplements like fetal bovine serum, fetal calf serum, human AB serum, and another commercially available platelet lysate preparation.
  • FIG. 10 is a graph plotting the typical amounts of the indicated growth factors found in manufactured platelet lysates. Eleven manufactured platelet lysate products (Production PL) were measured for the indicated growth factors by ELISA. The growth factor and the axis it uses are indicated below the columns. The amount of the indicated growth factors present within fetal bovine serum (FBS) was also measured.
  • FBS fetal bovine serum
  • FIG. 16 contains a graph plotting the cell proliferation potential and presence of PDGF in PL fractionated using molecular weight cut-off filters.
  • Cell proliferation calculated the number of cells after 800 cells/well were plated and grown for four days containing the indicated component and a graph plotting the amount of PDGF-BB (ng/mL) measured by ELISA in the indicated component.
  • HCTL PL 5% PL using advanced MEM as the base media.
  • FIG. 17 contains a graph (top) plotting the A 280 for the indicated fractions of PL isolated by size fractionation and a photograph (bottom) of a silver stained reducing gel containing the indicated fractions.
  • FIG. 18 is a scanning electron photograph of complexes present with a platelet lysate produced as described in Example 2.
  • FIG. 19 is a graph plotting the amount of the indicated cytokine or growth factor present within platelet lysates.
  • FIG. 20 contains four graphs comparing the levels of PDGF-BB, FGF, TGF- ⁇ , and IGF-1 present with platelet lysates produced as described in Example 2 (HCTL production) to the levels of PDGF-BB, FGF, TGF- ⁇ , and IGF-1 reported in the indicated references or bovine serum.
  • Lange et al. is the Lange et al. reference ( Cellular Therapy and Transplantation, 1(2) December (2008)); Weibrich et al. is the Weibrich et al. reference ( Craniomaxillofac. Surg., 30:97-102 (2002)); and Doucet et al. is the Doucet et al. reference ( J. Cell Physiology, 205: 228-236 (2005)).
  • FIG. 21 is a graph plotting relative proliferation (absorbance) for MSCs cultured as indicated.
  • FIG. 22 is a graph plotting the cell count for adult stem cells cultured for the indicated number of days in medium (alpha-minimal essential medium (a-MEM)) supplemented with 5 percent of a platelet lysate produced as described in Example 2 or medium supplemented with 10 percent fetal calf serum.
  • medium alpha-minimal essential medium (a-MEM)
  • FIG. 23 is a graph plotting the population doublings for MSCs cultured in the indicated media.
  • FIG. 24A is a table listing the results from culturing primary tumor cells with the indicated media
  • FIGS. 24B , 24 C, 24 D, and 24 E contain data regarding GBM103, GBM 106, GBM 110, and GBM 111 cell cultures, respectively.
  • FIG. 25 contains four graphs plotting the number of cells at the indicated day for primary tumor cells grown in PL (AIM-V media supplemented with 5% platelet lysate as per example 2), NSC media (Neurobasal-A media with N2 and B27 supplements and EGF and FGF), or DMEM 10% FBS.
  • PL AIM-V media supplemented with 5% platelet lysate as per example 2
  • NSC media Neuroblastsal-A media with N2 and B27 supplements and EGF and FGF
  • DMEM 10% FBS DMEM 10% FBS.
  • FIG. 26 is a graph plotting the percent positive dendritic cells for the indicated marker of CD14+ monocytes cultured ex-vivo with 1% HABS in X-Vivo 15 media (XV-HABS), 1% platelet lysate-containing X-Vivo 15 media (XV-PL), or CellGro serum free media (no supplementation). All indications contained 2800 IU GM-CSF and 1000 IU IL-4 for the entire course of culture (six days). All conditions had TNF- ⁇ and PGE2 added on day three, and the cells were phenotyped on day six. For XV-PL (3-6) the culture was incubated in HABS for the first three days and PL for the last two.
  • FIG. 27 is a graph plotting the total MSCs after five days of culture with the indicated compositions.
  • FIG. 28 Effect of culture condition on cell adherence. Tumors (in rows) were physically and enzymatically digested and incubated in three culture conditions (NSC, neural stem cell; PL, platelet lysate; or FBS, fetal bovine serum). Cells were photographed and descriptions of the morphology listed.
  • NSC neural stem cell
  • PL platelet lysate
  • FBS fetal bovine serum
  • FIG. 29 PL promotes rapid growth of primary GBM cultures.
  • FIG. 30 is a graph plotting cell growth observed with the indicated amount of platelet lysate (PL).
  • FIG. 31 Presence of PDGF and FGF in PL after 48 days of incubation at different temperatures. PL samples were incubated at the temperatures identified for four different lots of PL. Growth factors were measured by ELISA. Each dot represents the mean of three samples from that lot incubated at that temperature. Data plotted is the means from each of these lots. Bars are the mean of four lots, with changes represented by asterisk (p ⁇ 0.05 for paired samples).
  • FIG. 32 is a graph plotting the amount of PDFG and FGF in PL samples incubated at the indicated temperatures.
  • FIG. 33 is a graph plotting the amount of PDFG in PL samples incubated at the indicated temperatures.
  • FIG. 34 is a graph plotting the PDFG decay rate.
  • FIG. 35 is contains the karyotype data for two mesenchymal stromal cell isolates cultured in either 10% FBS or 5% PL.
  • compositions containing platelet contents e.g., platelet lysates and/or supernatants or filtrates from platelet-containing media.
  • this document relates to methods and materials for making and using compositions containing platelet contents.
  • Any appropriate source of platelets can be used to make a composition containing platelet contents.
  • apheresis platelets and platelets derived from normal blood donation can be used as a source of platelets for making a platelet lysate composition, a supernatant or filtrate from a platelet-containing medium, or a platelet plasma culture supplement.
  • a composition containing platelet contents can be obtained as follows. Once obtained, platelets contents can be released using any appropriate method including, without limitation, a single freeze/thaw cycle, repeated (e.g., 2, 3, 4, 5, or more) freeze/thaw cycles, detergent lysis, activation with thrombin, collagen, thromboxane A2, ADP or other factors, and manipulation of ionic strength. In some cases, two freeze/thaw cycles can be used to obtain lysed platelets. Once lysed, the lysed platelet preparation can be centrifuged to obtain a supernatant.
  • a single freeze/thaw cycle repeated (e.g., 2, 3, 4, 5, or more) freeze/thaw cycles, detergent lysis, activation with thrombin, collagen, thromboxane A2, ADP or other factors, and manipulation of ionic strength.
  • two freeze/thaw cycles can be used to obtain lysed platelets. Once lysed, the lysed platelet preparation can be centrifuged
  • the force of centrifugation can be between 2000 ⁇ g and 5000 ⁇ g, and the duration can be between 10 minutes and 60 minutes.
  • a lysed platelet preparation can be centrifuged at about 3,000 ⁇ g for about 30 minutes. Once the supernatant is collected, it can be filtered. For example, the supernatant can be filtered through a 0.45 ⁇ m filter, a 0.2 ⁇ m filter, or a 0.45 ⁇ m filter followed by a 0.2 ⁇ m filter. The resulting filtrate can be used as a platelet lysate composition without further processing or can be combined with heparin to form a platelet lysate composition.
  • a platelet lysate provided herein can be prepared without washing the platelets prior to lysing them.
  • the platelet lysate can include plasma and plasma components.
  • a platelet lysate provided herein can include albumin and/or thrombin at about physiologic concentrations.
  • a platelet lysate provided herein can include platelet contents prepared from platelets lysed in the presence of plasma or a plasma-like composition.
  • a composition containing platelet contents can be obtained as follows.
  • Platelets can be maintained between 2° C. and 42° C. (e.g., between 2° C. and 40° C., between 2° C. and 38° C. between 2° C. and 36° C., between 2° C. and 30° C., between 5° C. and 36° C., between 10° C. and 36° C., between 15° C. and 36° C., between 20° C. and 30° C.) for a period of time (e.g., two, three, four, five, or more days) in the presence of plasma without performing an active step designed to lyse the platelets.
  • a period of time e.g., two, three, four, five, or more days
  • a platelet preparation obtained from an apheresis technique can be used without removing the plasma.
  • the platelet preparation can be treated to remove platelets, platelet debris, or platelet ghosts, while obtaining the resulting medium that includes platelet contents and plasma components.
  • this resulting medium can be obtained by centrifugation and/or filtration.
  • the resulting medium can be stored or used as a composition containing platelet contents or a platelet plasma culture supplement as described herein.
  • compositions containing platelet contents can be formulated with any appropriate medium to produce a culture medium having enhanced properties.
  • media that can supplemented with a composition provided herein include, without limitation, DMEM, RPMI, AIMV, X-VIVO15, and other defined serum free or serum requiring media.
  • Any appropriate amount of a composition containing platelet contents provided herein can be added to a medium.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more percent (e.g., vol/vol) of a medium can be a platelet lysate composition or a platelet plasma culture supplement provided herein.
  • Such supplemented media can be used to promote enhanced growth of cells (e.g., normal adult progenitor cells, stem cells, precursor cells, fibroblasts cells, and mesenchymal stromal cells), to differentiate progenitor cells, stem cells, and precursor cells into functional subtypes efficiently, to promote enhanced growth of tumor stem cells in primary tumor cultures, to establish tumor cell lines, or to generate primary cell cultures (e.g., tumor cell lines) efficiently.
  • cells e.g., normal adult progenitor cells, stem cells, precursor cells, fibroblasts cells, and mesenchymal stromal cells
  • compositions containing platelet contents can be used to obtain superior growth kinetics when culturing cells (e.g., progenitor cells and tumor stem cells) as compared to cultures using supplementation from human serum, fetal bovine serum, or serum-free media with recombinant growth factor supplementation.
  • compositions containing platelet contents can be used to supplement media used to culture cells from any species including, without limitation, humans, monkeys, horses, dogs, cats, rats, or mice.
  • differentiated cells can be derived from mesenchymal stem cells by incubating the mesenchymal stem cells with a composition (e.g., culture media) containing one or more factors together with a composition containing platelet contents (e.g., a platelet lysate composition) provided herein.
  • a composition e.g., culture media
  • platelet contents e.g., a platelet lysate composition
  • the factors can be any type of factors such as polypeptides, steroids, hormones, and small molecules. Examples of such factors may include, without limitation, dexamethasone, EGF, FGF and BMP4.
  • Stem cells can be incubated with a such compositions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 days.
  • a composition provided herein and used to promote cell growth or differentiation can be replaced every day or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more days.
  • stem cells e.g., mesenchymal stem cells
  • the state of differentiation can be monitored to determine whether or not the stem cells differentiated into differentiated cells having a desired phenotype.
  • a sample of cells can be collected and assessed using techniques such as Western blotting, fluorescence-activated cell sorting (FACS), immunostaining, laser confocal microscopy, and reverse transcription polymerase chain reaction (RT-PCR) techniques (e.g., quantitative RT-PCR).
  • FACS fluorescence-activated cell sorting
  • RT-PCR reverse transcription polymerase chain reaction
  • this document also provides isolated cell cultures.
  • this document provides isolated primary tumor cell cultures such as a GBM 106 cell culture or any other tumor cell culture identified in FIG. 24A .
  • the isolated primary tumor cell cultures identified in FIG. 24A can be designated with an “MC” prefix when established using a composition containing platelet contents provided herein.
  • the GBM106 cell culture obtained using a platelet lysate provided herein can be designated as MCGBM106.
  • the GBM110 cell culture obtained using a platelet lysate provided herein can be designated as MCGBM110, and so on.
  • An isolated primary tumor cell culture provided herein can be obtained from a human patient and cultured in the presence of medium supplemented with a composition containing platelet contents.
  • a medium containing between 1 percent and 50 percent e.g., between 1 percent and 45 percent, between 1 percent and 35 percent, between 1 percent and 25 percent, between 1 percent and 15 percent, between 1 percent and 10 percent, between 1 percent and 5 percent, between 5 percent and 50 percent, between 10 percent and 50 percent, between 20 percent and 50 percent, between 3 percent and 10 percent, or between 3 percent and 7 percent
  • a platelet plasma culture supplement provided herein can be used to culture human primary glioma cells.
  • Cells (e.g., primary tumor cells) cultured as described herein can exhibit rapid growth kinetics ( FIGS. 1 and 29 ), higher frequency of cell line establishment ( FIGS. 24A-E ), and stable genetics ( FIG. 1 ).
  • FIG. 1 it is noted that the karyotype of cells cultured with the HCTL#3 and the NSC media can be very similar, while the karyotype of the cells cultured with FBS can be different. This was done starting with a single tumor sample. Thus, the FBS cultured cells evolved away from the other two cell lines. The other two cell lines are very similar suggesting that these two reflect the progenitor tumor.
  • primary tumor cells cultured as described herein can exhibit a constant doubling time ( FIG. 29 ).
  • a constant doubling time can indicate that there is no significant lag time in establishing the culture. Thus, there are no other needed factors that require genetic rearrangements/or additives to have the cultures grow.
  • Primary tumor cells cultured as described herein also can exhibit the presence of cells with progenitor phenotypes.
  • An example of SOX2 and CD 133 expression in a primary tumor culture is provided in FIG. 1 .
  • SOX2 and CD133 are markers of progenitor cells associated with tumor progenitor cells (tumor stem cells).
  • Primary tumor cells cultured as described herein can be GMP compatible since the supplement (e.g., a composition containing platelet contents such as a platelet plasma culture supplement) can be GMP compatible.
  • primary tumor cells cultured as described herein can be biologically relevant in that many tumors are associated with blood clots. While not being limited to any particular mode of action, a blood clot may act like an in vivo matrix for the collection of platelet supernatants. Thus, a composition containing platelet contents provided herein can mimic the normal biology of the tumor.
  • UDQ Uniform Donor History Questionnaire
  • This questionnaire is a screening document created by a coalition of regulatory, accrediting, and blood collecting institutions consisting of the Food and Drug Administration, Centers for Disease Control and Prevention, Armed Services Blood Program, National Heart Lung and Blood Institute, American Blood Resources Association, AABB, American Red Cross, and America's Blood Centers. Information concerning the UDQ can be found on the World Wide Web at “fda.gov/cber/dhq/dhq.htm.”
  • All apheresis platelet donations were tested with the following infectious disease tests: (1) Serologic test for syphilis; (2) HCV EIA-hepatitis C virus antibody test, (3) HCV NAT-hepatitis C virus nucleic acid test, (4) HbsAg-hepatitis B surface antigen test, (6) Anti-HBc-hepatitis B Core antibody, (7) HIV-1/2 EIA-Human Immunodeficiency Virus 1/2 antibody test with ability to detect HIV 1 subgroup O; (8) HIV NAT-Human Immunodeficiency Virus nucleic acid test, (9) HTLV I/II EIA-Human T-Lymphotrophic Virus Types I/II, (10) WNV NAT-West Nile Virus nucleic acid test, and (8) Anti-T. cruzi, (serologic test for Chagas disease) using FDA licensed procedures.
  • the platelet products that were released for manufacture of the growth media were collected from donors who fulfill donation criteria, were negative tests for the infectious diseases listed above, and exhibited no evidence of bacterial growth by their expiration date. Products from donors who failed to meet donor criteria, that exhibited positive infectious disease testing, or that produced cultures positive for bacteria were considered biohazardous waste. These products were quarantined and destroyed. They were not released for manufacture of a platelet lysate growth media. FDA tests and guidelines for release can change. However, platelets used for these purposes met FDA tests and guidelines current at the time of production.
  • Apheresis platelets were obtained as described in Example 1.
  • the apheresis platelets used were no more than four days past expiration.
  • a single lot of platelet lysate consisted of ten individual apheresis platelet units, and one lot was used at a time to create a platelet lysate product.
  • the processing for clinical grade reagents can be performed in a clean room suite.
  • Ten individual apheresis platelet units were frozen at ⁇ 70° C. or colder. After being frozen for at least 24 hours, the units were removed from the freezer and allowed to thaw. The units were thawed at room temperature or at refrigerated temperatures. After thawing was complete, each unit was mixed by massaging the bag.
  • Each thawed platelet bag was placed flat (to minimize breakage of tubing) in a supercold freezer ( ⁇ 70° C. or colder) for a second freeze. After the apheresis platelet units were frozen for at least 24 hours for a second freeze, they were removed from the freezer and allowed to thaw. After the second thaw, the platelet product was aseptically transferred to 250 mL conical centrifuge tubes. The tubes were centrifuged for 30 minutes at 3000 ⁇ g for 30 minutes at room temperature using a Benchtop Centrifuge Sorvall Legend T. The resulting supernatants were transferred to 0.45-micron filter units (Pall Stericup, Catalog Number SCHV U05 RE; East Hills, N.Y.
  • Nalgene Filter System Catalog Number 167-0045; Rochester, N.Y.
  • pre-filters Glass Microfibre filters GF/B or GF/D used interchangeably; Whatman®, Florham Park, N.J.
  • the filter unit was connected to a vacuum source and allowed to filter the product. If the product did not filter completely, the unfiltered product was transferred to another filter unit with a pre-filter.
  • the filtrates from all of the 0.45-micron filter units were pooled and filtered through a 0.2-micron filter unit (Pall Stericup, Catalog Number SCHV U05 RE; East Hills, N.Y.
  • Nalgene Filter System Catalog Number 567-0020; Rochester, N.Y.
  • pre-filters Glass Microfibre filters GF/B or GF/D used interchangeably; Whatman®, Florham Park, N.J.
  • the filter unit was connected to a vacuum source and allowed to filter the product. If the product did not completely filter, the unfiltered product was transferred to a second filter unit, and the process was repeated as needed. The 0.2-micron filtrates were combined into receiver bottles.
  • a plasma transfer set was connected to a 60-mL syringe, and the transfer set was spiked into a 2 L blood bag. Using the syringe as a funnel, the filtered lysates from the apheresis platelet units were combined into the 2 L bag. The contents were mixed well. Heparin (1000 U/mL) was added to the filtered platelet lysates to obtain a final concentration of two U/mL.
  • the lysates were divided into aliquots.
  • the lysates were stored frozen at ⁇ 20° C. or colder.
  • Endotoxin Assay One mL of platelet lysate was transferred to a sterile endotoxin-free tube that was used to perform an endotoxin assay. Briefly, a 1:50 dilution of Platelet Lysate to Limulus Amebocyte Lysate (LAL) Reagent Water was run on the Endosafe Portable Test System (PTS; Charles River, Wilmington, Mass.). The Endosafe PTS utilizes LAL kinetic chromogenic methodology to measure color intensity directly related to the endotoxin concentration in a sample. Each disposable cartridge contains precise amounts of licensed LAL reagent, chromogenic substrate, and control standard endotoxin. The result obtained from each batch of Platelet Lysate must be ⁇ 0.500 Endotoxin Units (EU)/mL.
  • EU Endotoxin Units
  • the supernatant was removed from the tube, and one mL of the PL5% media was added to the cell pellet. A cell count was performed.
  • the thawed cells were placed in one to two 175 cm 2 flasks with 50 mL of PL5% so that each flask contained 1.75 ⁇ 10 5 -4.38 ⁇ 10 5 of the thawed cells.
  • the flasks were incubated at 37° C. in a 5 percent CO 2 incubator.
  • the cells were passaged using TrypLETM (Invitrogen Corporation, Carlsbad, Calif.) after the flasks were confluent.
  • the cells were combined, and a cell count performed. A population doubling calculation was performed.
  • Platelet lysates that were sterile, endotoxin-free, grew MSC with the expression profile of CD105, CD90, CD73, HLA-ABC positive and negative for CD14, CD45, and HLA-DR were released for clinical use and assigned an expiration date of two years from production.
  • the phenotypic characteristics of GBM cells cultured with media containing platelet lysates were compared to those of GBM cells cultured using NSC media (Neurobasal media (Invitrogen, Grand Island N.Y.); recombinant EGF and FGF (R&D Systems, Minneapolis, Minn.), N2 and B27 supplements (Invitrogen, Grand Island N.Y.), glutamine and penicillin/streptomycin) or DMEM containing 10% FBS.
  • the media containing platelet lysate (HCTL#3) consisted of Neurobasal media supplement with 5% platelet lysate, glutamine, and penicillin/streptomycin.
  • Cells grown in NSC media formed classical neurospheres enriched in tumor stem cells ( FIG. 1A ).
  • FIG. 1A Cells grown in DMEM 10% FBS became adherent and were usually differentiated.
  • FIG. 1B Cells grown in HCTL#3 resulted in a mixed population of free-floating neurospheres and adherent neuropheres with superior growth kinetics.
  • FIG. 1C Cells cultured in HCTL#3 also exhibited many aspects of neural tumor stem cells and were positive for CD133, nestin, and SOX2 ( FIG. 1C ).
  • FIG. 1D Kitange et al., Curr. Opin. Oncol., 15(3):197-203 (2003)).
  • FIGS. 24A-E and 25 Additional results culturing primary tumor cells are presented in FIGS. 24A-E and 25 . These additional results demonstrate that the platelet lysates provided herein can perform better than fetal calf serum and a defined supplemented media (NSC) for growing primary tumor cells (e.g., gliomas) ( FIGS. 24A-E ). These results also demonstrate that platelet lysate-containing media can grow tumors in less time ( FIG. 25 ).
  • NSC fetal calf serum
  • FIGS. 24A-E primary tumor cells
  • Tumor cells grown in media supplemented with manufactured platelet lysate were enriched for primary GBM-associated tumor antigens that were similar, if not superior, to cells grown in NSC media ( FIG. 2 ).
  • the PL supplemented media allowed for the generation of primary cell cultures from GBM patients that exhibited superior growth kinetics, characteristic tumor genetic rearrangements, populations of neural tumor stem cells, and enrichment in certain tumor antigens.
  • the use of lysates from autologous tumor cultures as antigen for DC immunotherapy offers the advantage of targeting multiple tumor antigens reflective of the patient's antigen profile, eliminating contaminating cells, and providing sufficient antigen for multiple immunizations.
  • Preliminary research in GBM patients demonstrated the consistent ability to generate primary tumor cultures from small portions of resected tumor.
  • Neurobasal media Invitrogen, Grand Island, N.Y. plus 5% fully manufactured platelet lysate (platelet lysate), Neurobasal media containing 5 percent platelet lysate from a single freeze/thaw cycle without further processing (Min. Manipulated), Neurobasal media containing 5 percent human AB serum (HABS, Sigma, St.
  • MSCs were thawed and grown in Advanced MEM (Invitrogen, Grand Island, N.Y.) plus 5% HCTL manufactured platelet lysate until confluent. The cells were then passaged and split into Advance MEM with the following supplements: 5 percent (vol/vol) of a platelet lysate prepared as described in Example 2, platelet poor plasma (plt. poor plasma), whole platelets (whole plts.), or platelet exposed to two freeze/thaw cycles without further processing (Freeze/thaw plts.). The 5 percent platelet lysate was used as baseline with the others being adjusted to provide an equivalent protein concentration as the 5 percent platelet lysate.
  • Advanced MEM Invitrogen, Grand Island, N.Y.
  • HCTL HCTL manufactured platelet lysate until confluent.
  • the cells were then passaged and split into Advance MEM with the following supplements: 5 percent (vol/vol) of a platelet lysate prepared as described in Example 2, platelet poor plasma (
  • the cells were allowed to equilibrate in their specific media for a few days before beginning the experiment. Once prepared, the MSC were plated at 2.5 ⁇ 10 4 cells/plate into T25 flasks with their appropriate medium. The total number of cells in each of three flasks for each condition was counted on days three, four, and five ( FIG. 5 ). These results demonstrate that MSCs grew the best in HCTL fully manufactured lysate.
  • a cell line from a primary tumor was thawed and plated similarly to the MSC growth experiment. Conditioned cells were then plated at 8 ⁇ 10 4 cells/plate in Neurobasal Media (Grand Island, N.Y.) supplemented with either 5 percent (vol/vol) of a platelet lysate (PL) prepared as described in Example 2, platelet poor plasma (plt poor plasma or PPP), whole platelets (whole plts or WP), or platelet exposed to two freeze/thaw cycles without further processing (Freeze/thaw plts or FT). The 5 percent platelet lysate was used as baseline with the others being adjusted to provide an equivalent protein concentration as the 5 percent platelet lysate.
  • PL platelet lysate
  • the cells were allowed to equilibrate in their specific media for a few days before beginning the experiment.
  • the total number of cells in each of three flasks for each condition was counted on days three, six, and nine ( FIG. 6 ). These results demonstrate that cells grew better in fully manufactured platelet lysate over 6 days.
  • the platelet preparations included (1) minimally manipulated (MM) apheresis platelets from expired donations that were not frozen (3 bags were combined), (2) platelet poor plasma (PPP), which was a fraction of minimally manipulated apheresis platelets that were spun down at 3000 g for five minutes and then spun at 10,000 g for one minute, (3) a freeze/thaw fraction (FT), which contained minimally manipulated apheresis platelets that were subjected to two freeze/thaw cycles, (4) platelet lysate (PL) prepared as described in Example 2.
  • MM minimally manipulated
  • PPP platelet poor plasma
  • FT freeze/thaw fraction
  • PL platelet lysate
  • the MM, PPP, FT, and PL preparations were obtained from the same starting material and were stored for over two months at 4° C.
  • the PL preparation exhibited the highest level of VEGF ( FIG. 8 ).
  • VEGF and PDGF are consistent across several different lots of platelet lysate manufactured in the HCTL (14244, 14453, 14569, and 14593) ( FIG. 9 ).
  • these growth factors are significantly higher on a per volume basis than that of other growth supplements including fetal bovine serum, fetal calf serum, human AB serum, and another commercially available platelet lysate preparation (Cat. No. PNP-10, Lot No. PL14, Precision Biologic, Dartmouth, Nova Scotia).
  • PL were used to initiate primary tumor cultures derived from surgical specimens obtained from patients with renal cell carcinoma.
  • EGF, FGF, IGF-1, PDGF-BB, TGF- ⁇ , and VEGF present as measured by ELISA were analyzed to determine the amount of EGF, FGF, IGF-1, PDGF-BB, TGF- ⁇ , and VEGF present as measured by ELISA.
  • the measured amounts were compared to those measured in one or more of the following: fetal bovine serum (FBS), human AB serum (HABS), a commercially available platelet lysate (Cryocheck; catalog number PNP-10; Precision BioLogic, Inc., Nova Scotia, Canada) and fresh plasma without platelets.
  • FBS fetal bovine serum
  • HABS human AB serum
  • a commercially available platelet lysate Cryocheck; catalog number PNP-10; Precision BioLogic, Inc., Nova Scotia, Canada
  • the typical amounts of EGF, FGF, PDGF-BB, TGF- ⁇ , and VEGF within the manufactured platelet lots as compared to FBS are presented in FIG. 10 .
  • the average amount of FGF within the manufactured platelet lots was about 180 pg/mL, while the average amount present within a commercially available platelet lysate was 80 pg/mL ( FIG. 11 ).
  • the average amount of PDGF-BB within the manufactured platelet lots was about 8.5 ng/mL, while the average amount present within a commercially available platelet lysate was 2.9 ng/mL ( FIG. 12 ).
  • the average amount of IGF-1 within the manufactured platelet lots was about 132 ng/mL, while the average amount present within a commercially available platelet lysate was 7.7 ng/mL ( FIG. 13 ).
  • the average amount of TGF- ⁇ within the manufactured platelet lots was about 54 ng/mL, while the average amount present within a commercially available platelet lysate was 37 ng/mL ( FIG. 14 ).
  • the levels of PDGF-BB, FGF, TGF- ⁇ , and IGF-1 present in platelet lysates produced as described in Example 2 were compared to the levels of PDGF-BB, FGF, TGF- ⁇ , and IGF-1 reported in the literature or bovine serum ( FIG. 20 ).
  • the total amount of protein within eleven lots of platelet lysates manufactured as described in Example 2 was measured using a modified Bradford assay.
  • the average amount of total protein within the manufactured platelet lots was about 49 mg/mL, while the average amount present within the commercially available platelet lysates was 0.03 mg/mL ( FIG. 15 ).
  • the total amount of protein within the manufactured platelet lots was similar to the amounts measured in the fresh plasma without platelets, the fetal bovine serum, and the human AB serum ( FIG. 15 ).
  • the mean of the levels of growth factors present with the platelet lysates provided herein was about 2 to 3 times that of the commercially available platelet lysate.
  • the commercially available platelet lysate was not different than fetal bovine serum.
  • the platelet lysates provided herein had about 3000 times the amount of protein than that of the commercially available platelet lysates, yet protein alone cannot account for the activity as the platelet lysates provided herein since they exhibited similar levels of total protein as that found in human AB serum.
  • platelet lysates were produced from an outdated platelet concentrate from a single donor. The number of platelets for each lysing condition was equal, and the volume used for lysing was equal for all modifications and equal to the volume of the platelet concentrate used for the unmodified condition.
  • the platelet lysates were filtered on size selection filters with specific molecular weight cut-offs. The filtrate (less than the size indicated) and the retentate (greater than the size indicated) were analysed for the presence of PDGF-BB by ELISA and the ability to stimulate cell proliferation.
  • PDGF-BB has a molecular weight of about 30 KDa (30,000 MW protein).
  • PDGF-BB was observed only in the retentates including the >100,000 preparations suggesting that all of the PDGF-BB is bound to complexes greater than this size. Also, proliferation occurred in cultures containing these larger (>100,000 Da) complexes.
  • a platelet lysate (0.5 mL), which was thawed, centrifuged at 3,000 ⁇ g, and filtered through a 0.2 mm filter, was applied to a 1.5 cm ⁇ 27 cm column packed with Sephadex G-150-120 (Pharmacia Fine Chemical Company) and equilibrated with 50 mM sodium phosphate, 150 mM NaCl, pH 7.4 buffer. The chromatography was developed at a flow rate of 25 mL/hour, and the A 280 of each fraction was measured.
  • a sample from platelet lysate manufactured as described in Example 2 was examined using a scanning electron microscope. As shown in FIG. 18 , several large complexes are readily observable. Many particles were observed up to 0.2 ⁇ m in size.
  • MSCs were thawed and grown in a-MEM with 5% PL until confluent. The cells were then passaged and split into a-MEM medium only or a-MEM medium with the following supplements: 5 percent (vol/vol) Cryocheck, 10 percent (vol/vol) FBS, 5 percent (vol/vol) HABS, or 5 percent (vol/vol) of a platelet lysate prepared as described in Example 2. Relative proliferation was assessed using absorbance measurements. The greatest amount of proliferation was observed with MSCs cultured in the presence of the platelet lysate prepared as described in Example 2 ( FIG. 21 ).
  • adipose tissue MSCs were observed to grow faster in the presence of 5 percent of a platelet lysate produced as described in Example 2 than in 10 percent fetal calf serum ( FIGS. 22 and 23 ).
  • PL was used as a supplement instead of human AB serum in culture conducive to the differentiation of CD14+ monocytes into the immune stimulating cells dendritic cells (described in Dietz et al., Transfusion, 46(12):2083-9 (2006)).
  • PL were incubated for the entire culture condition or for the last stage (day 3-6) of dendritic cell culture characteristic of generating active mature dendritic.
  • the changes associated in the culture were measured with particular emphasis on the expression of CD80 and CD83; two characteristic markers of dendritic cell culture. No effect of the PL as a supplement was observed in these conditions. Thus, PL appears to have a benign effect on the immune response.
  • MSCs were plated in media, and cell growth was determined by counting.
  • the media supplements were as follows: (a) none (media only), (b) PL #1 (platelet lysate manufactured in the presence of plasma), (c) washed PL #1+plasma (platelet lysate manufactured after removal of plasma followed by adding back plasma), (d) PL #1 washed (platelet lysate manufactured after removal of plasma), (e) PL2+Benzamidine (platelet lysate manufactured in the presence of plasma plus the clotting inhibitor benzamidine), and (f) washed platelets (PC2) in buffer (platelets manufactured in the absence of plasma).
  • GBM Patient-derived primary glioblastoma multiforme
  • the culture medium described herein containing platelet contents is used for a highly efficient establishment of tumor cell cultures.
  • the medium is likely more relevant to tumor growth than other methods and can be optimized for clinical-scale production compliant with Good Manufacturing Practice (GMP) requirements.
  • GMP Good Manufacturing Practice
  • the effect of media supplementation with platelet contents on tumor culture was tested by splitting primary tumors and incubating them in one of three culture methods.
  • the first culture included 10% fetal calf serum (FCS or FBS) in culture media.
  • FCS or FBS fetal calf serum
  • the second culture contained culture media used to culture tumor stem cells (Neural stem cell media, NSC; Piccirillo and Vescovi, Expert Opinion on Biological Therapy, 7(8):1129 (2007); Fan et al., Seminars in Cancer Biology, 17(3):214 (2007)).
  • the third culture media contained media supplemented with 5% human platelet derived media supplement (platelet lysate, PL).
  • FIG. 28 The effect of media supplementation was clear ( FIG. 28 ).
  • FBS cultures were uniformly adherent monolayers, with NSC media mostly having spheres or non-adherent cells.
  • PL cultures existed within the complete spectrum of cell morphology. Cells continued in culture and were counted at each passage. PL was superior to other methods in the ability to generated large numbers of cells ( FIG. 29 ). Importantly, cells grown in PL demonstrated a constant growth rate for more than 10 doublings in the absence of an initial lag phase, suggesting an immediate proliferation response upon plating ( FIG. 29 , left panel).
  • PL induced immediate and profound proliferation in many primary tumor cultures.
  • a 56% success rate of establishing primary cell growth was obtained, and a 44% rate of generating greater than 30 million cells in 60 days was obtained.
  • Not a single culture grown in FBS could meet the latter requirement.
  • Thirteen percent of the tumors grown in NSC conditions met these criteria.
  • PL were greater than 3 times better at establishing primary tumor cultures than the next best method.
  • FBS established cell cultures are considered to have lost the undifferentiated nature of the in vivo tumor.
  • nestin, Sox2, and CD133 was measured in PL cultured cells. The majority of cells in all cultures were nestin and Sox2 positive.
  • Four of the seven cultures tested for CD133 expression had levels above background with one culture having more than 30% of the cells positive for CD133.
  • PL adipose derived mesenchymal stem cells from non-malignant donors were incubated in PL supplemented media. No abnormal karyotypes were found in up to twelve passages of cells. Thus, PL maintained the genetic integrity of normal cells in culture. GBM cultures were karyotyped and found to maintain a stable karyotype despite massive cell doublings (see, e.g., GBM106; FIG. 24C ). In some cases, cultures consisted of stable subclones. The tumor cultures exhibited characteristic deletions (such as pten) and expressed tumor antigens such as EGFr, survivin, and IL13-r. Taken together, these results suggest that PL is a highly effective growth supplement for the establishment and proliferation primary tumor cultures.
  • the methods and materials provided herein can allow to the generation of autologous tumor cells for use in patient specific vaccines, or other immune modulatory protocols where patient specific material may be required.
  • the efficient in vitro generation of tens of millions of cells from >40% of all GBM tumors can allow for many new approaches to cancer treatment, including (a) the possibility to generate sufficient patient specific materials for repeated vaccinations for immune therapy protocols, (b) the establishment of a primary tumor cell library for drug screening on cell cultures having substantially fewer passages and more relevant material, (c) the development of specific drug screening protocols for patient specific optimization of chemotherapy, and (d) the cataloguing of the molecular profile of these primary tumor cultures and the association of these profiles with chemo-sensitivity.
  • a clinical trial is performed to confirm the use of autologous primary GBM cell cultures, which are produced as described herein, as an antigen source for vaccine studies.
  • Primary cell cultures are established from material obtained at surgery. Patients undergo standard care.
  • the patient begins vaccinations using the primary cell cultures combined with immune stimulating adjuvants.
  • Possible adjuvants for a tumor vaccine include dendritic cells, alum, GM-CSF, LPS, KLH etc. as well as other promising adjuvants.
  • the generated cGMP material is used directly as tumor antigen for allogeneic donors. Patients have their tumors molecularly matched to the corresponding library, a match is identified, and is subsequently used for treatment.
  • the cell cultures produced as described herein are screened for drug-mediated suppression of cell growth using compounds from the NIH clinical collection.
  • This collection represents drugs used in early phase clinical trials with described adequate safety profiles and commercial availability.
  • Drugs identified as candidates for cell suppression are tested in vivo on the same tumors growing in immune deficient mouse models.
  • drugs identified as candidates are matched to the transcription profile or if the patient cells are growing fast enough, drugs tested on the patient material and used on the patient allowing patient specific drug screening and use.
  • Platelet lysate was manufactured as described in Example 2. Immediately after manufacture, PL was aliquotted into multiple vials and transferred to ⁇ 70° C., 5° C., 25° C., or 37° C. for long-term storage. Sufficient aliquots were transferred such that samples were maintained at a single temperature until analysis. Thus, the data represent samples stored at a constant temperature for the time indicated. Three independent lots of PL were analyzed for the presence of growth factors and two for the ability to proliferate MSC.
  • MSC Proliferation Adipose derived, adult mesenchymal stromal cells (MSC) were obtained from fat collected as waste from gastric by-pass surgery. Fat was processed, and MSC were expanded and frozen until use in these assays. For these assays, MSC were thawed and allowed a single passage prior to plating for analysis. Growth was determined using the BioVision Quick Cell Proliferation Assay Kit II (Mountain View, Calif.). This assay is based on the cleavage of tetrazolium salt to formazin by cellular mitochondrial dehydrogenase. The amount of dye cleaved is proportional to live cells.
  • MSC were plated at 1 ⁇ 10 4 cells per well in RPMI1640 containing the designated amount of PL from the indicated conditions. The cells were incubated for 48 hours in 37° C., 5% CO 2 humidified incubator. The detection reagent was added (10 mL of WST) to each well. The plate was incubated for one hour, and the O.D. measured per manufacturer's directions.
  • Growth Factors An ELISA test on PL that was stored at the indicated temperatures was used to determine the concentration of the growth factors of interest. All ELISAs were from R&D Systems (Minneapolis, Minn.) and used according to manufacturer's instructions.
  • MSC Long-term storage of PL at 37° C. reduced, but did not eliminate, MSC growth ( FIG. 30 ). MSC were cultured with different concentrations of PL that had been maintained at ⁇ 70° C. or 37° C. for 48 days. There was a typical dose response curve observed for both PL incubated at ⁇ 70° C. and 37° C. Interestingly, the PL incubated for this long at body temperature maintained its ability to stimulate 70% of the expected growth of PL at a similar concentration.
  • the levels of FGF and PDGF were measured in four lots of PL held at ⁇ 70° C., 5° C., 25° C., and 37° C. ( FIGS. 31 and 32 ). These concentrations were measured immediately after production, and on days 7, 14, 21, and 48.
  • PDGF was the most sensitive to the effects of accelerated aging. Therefore, the shelf life results were derived from that data ( FIG. 33 ).
  • the slope intercept method (Arrhenius equation), Q rule, and conservative approaches were used to estimate the shelf life of platelet lysates ( FIG. 34 and Table 1).
  • compositions provided herein containing platelet contents have an estimated shelf life of greater than five years at ⁇ 70° C. and an estimated shelf life of about one year at ⁇ 20° C.

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US20120231542A1 (en) * 2011-03-11 2012-09-13 General Biotechnology, Llc Biologically Active Human Umbilical Cord Blood Cell Extract Compounds and Methods
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WO2023193699A1 (zh) * 2022-04-06 2023-10-12 上海我武干细胞科技有限公司 血清替代物及其制备方法与应用
CN114836381A (zh) * 2022-05-22 2022-08-02 广州捷创生物科技有限公司 诱导间充质干细胞定向分化为神经细胞的方法及其培养基

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