WO2007149000A2

WO2007149000A2 - New structural protein preparations

Info

Publication number: WO2007149000A2
Application number: PCT/PL2007/000041
Authority: WO
Inventors: Andrzej Lipkowski; Marcin Jurga; Krystyna Domanska-Janik; Barbara Lukomska
Original assignee: Instytut Medycyny Doswiadczalnej I Klinicznej Im.
Priority date: 2006-06-23
Filing date: 2007-06-22
Publication date: 2007-12-27
Also published as: WO2007149000A3

Abstract

Novel proteinaceous skeleton preparations were described, devoid of proteins susceptible to mammalian proteolytic enzymes as well as a method of obtaining these preparations, particularly keratin-based, encompassing the use of repeated chemical activation, enzymatic digestion and grinding of the obtained keratinous preparations, as well as the application of the proteinaceous skeleton preparations, particularly as a carrier for use in stem cell cultures. Proteinaceous skeleton preparations resistant to enzymatic digestion may widespread find use in medicine as a carrier material for cell cultures, preferentially of stem cells.

Description

New structural protein preparations, their method of manufacture and applications

The subject of the present invention axe novel protein preparations for medical application as a skeletal micromaterial for culturing stem cells and a method of manufacturing such preparations, as well as their application in the stem cell cultures. The central and peripheral nervous systems form a an integrated network which regulates the functioning of entire human and animal organisms. Disruptions in its functioning are currently one of the most serious problems of modern medicine. Physical damage to the nervous system, such as the spinal chord, is often the cause of permanent crippling following automotive accidents or injuries resulting from extreme . sports. Neuropathological changes caused by tumor growth or autodegradation (Parkinson's and Alzheimer's diseases or Multiple Sclerosis) also lead to irreversible impairment. In contrast to other tissues, the nervous system regenerates poorly. The recently demonstrated ability of stem cells to transform into differentiated adult cells, including neurons, opens up possibilities of medically correcting neurpathologies resulting from physical or biological damage or senescence. At the same time, in vitro stem cell cultures destined for transplantation purposes require the use of a skeletal micromaterial facilitating cell growth in three dimensions.

Polish patent PL 179302 describes a method of obtaining an abrasive material for cleaning and exfoliating skin which can be used in the manufacture of protein preparations for use as skin exfoliants in cosmetics. The method according to said invention consisted of the enzymatic digestion of natural sources of structural proteins such as hair, bristles, wool or feathers and then grinding the resulting solid matter down to a particulate protein preparation of a pre-determined diameter. Research showed that the protein preparations obtained according to said patent do not cause allergies when used as an exfoliant in cream. Further research unexpectedly showed that a single enzymatic digestion of the mentioned natural raw materials according to the extant method is only partial and leaves a portion of the digestible proteins in undigested form. Due to the pulverisation of the material, the digestive enzymes gain access to digestible proteins to which they did not have during the digestion of hair, bristles, wool or feathers. It is conceivable that such a ground preparation used in biological conditions will lead to the release of said proteins in contact with living cells or tissues. When used medically, the released proteins may cause a series of undesirable reactions, including allergies.

The goal of the present invention is to propose a new method of obtaining an improved skeletal protein preparation, devoid of the above mentioned drawbacks. Unexpectedly, this goal has been achieved in the present invention.

Unexpectedly it was discovered that a second enzymatic digestion of the preparation obtained via known methods leads to the disappearance of a majority of soluble proteins to a degree facilitating their use as a skeletal protein material in stem cell cultures. The application of human hair as a raw material makes it possible to perform individualised therapy based on the use of stem cells cultured on a proteinaceous skeleton preparation obtained according to the given examples from a patient's own hair. The subject of the present invention jest is a carrier protein preparation, characterised in that it contains proteinaceous skeleton preparations produced from the epithelial products selected from among animal fur, bristles, wool and hair, particularly human hair, or avian feathers, preferentially porcine bristles or human hair. A preparation "essentially free of soluble proteins" is understood to be one according to the present invention, which loses no more than 0.5% of its dry mass upon repeated hydrolysis with any given enzyme.

Another subject of the present invention is also a method of obtaining a carrier protein preparation, characterised in that epithelial products of vertebrates are sequentially voided of proteins susceptible to enzymatic degradation, wherein during subsequent stages of pulverization they are activated with inorganic reagents, digestion with a protease and washing at least once, and the carrier protein preparation obtained is isolated. Equally preferentially, the activation of intermediary preparations is performed using sodium or potassium hydroxide at a concentration of 0.1% to 4% by mass. Equally preferentially, digestion with a proteolytic enzyme is performed using an enzymatic preparation containing an enzyme selected from among: pepsin, chymotrypsin, trypsin, pancreatin or any combination of the above. Equally preferentially, the isolated carrier protein preparation loses less than 0.5% of its dry mass when further enzymatic hydrolysis is attempted.

A further subject of the present invention is also the application of a carrier protein preparation according to the present invention or one obtained according to the present invention in the manufacture of a carrier for stem cells. Preferentially, the carrier produced is used in a stem cell culture as a skeleton substance for forming aggregates of stem cells; destined for further application in the regeneration of cells and tissues in the human. Equally preferentially, aggregates of cells derived from stem cells cultured on the protein material obtained are destined to be used as implants which rescue the functions of damaged cells of the nervous, muscular and endocrine systems. Equally preferentially, a patient's own hair is used to manufacture the carrier for a cell aggregate implant that patient is to receive. Equally preferentially, the carrier produced is used in the culture of stem cells without the use of animal sera or other culture ingredients of animal origin. The subject of the present invention is also a cell aggregate characterised in that it contains cells and a carrier protein preparation according to the present invention. Preferentially it contains stem cells or their derivatives.

"Stem cell-derived cells" are understood to be any given cells developing from stem cells, which may be found at various stages of differentiation, contained in the aggregate formed on the carrier containing the carrier protein preparation according to the present invention. The subject of a particular embodiment of the present invention are novel proteinaceous skeleton preparations obtained from hair, feathers, bristles or wool voided of proteins susceptible to digestion by mammalian proteolytic enzymes, to be used in medicine as a carrier material in stem cell cultures, as well as a method of obtaining novel proteinaceous skeleton preparations. Proteinaceous skeleton preparations devoid of proteins susceptible to digestion by mammalian proteolytic enzymes for medical use as a carrier material in stem cell cultures as well as a method of obtaining said proteinaceous skeleton materials according to the present invention consists of the application of a preparation obtained through the repeated alternating chemical activation and enzymatic digestion of proteinaceous skeleton preparations in a cell culture. To better illustrate the subjects of the present invention, novel proteinaceous skeleton preparations and the method of obtaining novel proteinaceous skeleton preparations and the method of manufacturing said proteinaceous skeleton preparations and the application thereof in stem cell cultures are presented in the Examples. The scope of the present invention should not be limited solely to the contents of the examples below. Example I. 1 kg of wool was submersed in 1% sodium hydroxide and stirred for 1 hour at room temperature. The lye was then drained and the wool was washed twice with water and submersed in water. The suspension of wool in water was acidified to a pH of 2.2 with HCl and pepsin was added. The mixture was stirred at a temperature of 35⁰C. The wool solids were drained off, washed twice and dried. The dry preparation was ground, and fines of less than 1 mm in length were collected. The preparation obtained was submersed in 1% lye and after 1 hour the product was drained off, washed in water and 1% hydrochloric acid. The remainder was resuspended in water, acidified to pH 2.2 and treated with pepsin. The mixture was incubated for 2 hours at 35⁰C, and then drained, washed three times in water and dried. 500 g of proteinaceous skeleton material were obtained from sheep's wool, designated SSP-2. The SSP-2 material obtained was added to a culture of human neural stem cells at a density of 10 million cells per millilitre. The culture was maintained under known conditions previously described for cultures of these stem cells without skeleton material. During the first day of culture on a medium containing 30% FBS, human neural stem cells divide and settle in even, multiple layers along the entire length of the proteinaceous skeletal material. Cells cultured on the proteinaceous material form durable conglomerates facilitating theirtransfer to other tissue cultures forming heterogenous cultures or for implantation into living tissue in vivo. Example II.

100 g of human hair was submersed in 1% lye and mixed for 1 hour at room temperature. The lye was then drained off and the hair was washed twice with water and submersed in water. The suspension of hah^* was acidified with hydrochloric acid to pH 2.2 and pepsin was added. The mixture was agitated for 3 hours at 35⁰C, and then the hair preparation was drained off and dried. The dry preparation was ground and sieved to collect fines less than 1 mm long. The preparation obtained was submerged in 1% lye. After 1 hour the product was centrifuged, washed with water, centrifuged, nd the remainder was washed in 1% hydrochloric acid and centrifuged. The remainder was suspended in water, the suspension was acidified to pH 2.2 and pepsin was added. The suspension was agitated for 2 hours at 35⁰C, drained, washed thrice with water and dried. The dried preparation was ground once more and sieved to collect product less than 0.25mm long. The dried preparation was submersed in 1% lye, and following an hour it was centrifuged, washed in water, centrifuged and the remainder was washed with 1% hydrochloric acid and centrifuged. The remainder was suspended in water, acidifed to pH 2.2 and pepsin was added. The suspension was agitated for 2 hours at 35⁰C, and then centrifuged. The insoluble fraction was washed through suspension in water and centrifugation, and then the insoluble remainder in water was dried. 25 g of proteinaceous skeleton material from human hair was produced, designated in short form HSP -2 .

Human neural stem cells cultured under conditions typical for media with serum were supplemented with the proteinaceous skeleton material from human hair, HSP-2. The culture was incubated under standard conditions (air with 95% humidity, 5% CO₂ at a temperature of 37⁰C). Twice a day, half of the medium was exchanged. The proliferating cells settled onto the proteinaceous skeleton material. The forming conglomerates of proliferating cells could be cultured for a month. The arising conglomerates could be used in the implantation of proliferating cells into tissue systems in vitro or directly into living organisms. Example III

1O g of hair were submerged in 1% lye and agitated for 1 hour at room temperature. The lye was then drained off, and the hair was washed twice in water and resuspended in water. The suspension of hair in water was acidified to pH 2.2, pepsin was added and the suspension was mixed for 3 hours at 35⁰C. The hair solids preparation was washed twice in water. The dry preparation was ground and sieved to collect product between 1 and 13 mm long. The sieved preparation was submerged in 1% lye, and following an hour the product was centrifuged, washed with water, centrifuged, and the remainder was washed with 1% hydrochloric acid and centrifuged again. The remainder was suspended in water, and the resulting suspension was acidifed to pH 2.2 and pepsin was added. The suspension was mixed for 2 hours at 35⁰C, and then drained off, washed thrice with water and dried. 2.0 g of proteinaceous skeleton material from human hair was produced, designated as HSP-3.

Human neural stem cells cultured in typical conditions in a medium without serum were supplemented with HSP-3 proteinaceous skeleton material from human hair. Further culturing was performed in an incubator under standard conditions (air at 95% humidity, 5% CO₂ at a temperature of 37 C). Half of the medium was changed twice per week. During the first week of culture in a medium without serum a single layer of cells settled onto the entire length of the suspended skeleton. During the subsequent 7 days, these cells formed local, multilayered concentrations around the central core of the skeleton. During the third and fourth week of culture, the local concentrations of cells joined up, forming a unified Podczas trzeciego i czwartego tygodnia hodowli lokalne skupiska komόrek lajczyly siς, tworzajc jednolity wielowarstwowy agregat , multilayered aggregate concentrically layed out on the central skeleton. The cells within the aggregate form gap junctions, and the cell layer reaches depths of 300 μm. The formed, floating conglomerates of cells on protein skeletons can be maintained in culture for at least another month without morphological changes. Over time, the internal skeleton is digested by the surrounding and infiltrating cells.

Claims

Patent Claims

1. A carrier protein preparation, characterised in that it contains proteinaceous skeleton preparations obtained from filamentous epithelial growths of vertebrates, particularly birds and mammals, essentially devoid of soluble proteins.

2. A protein preparation, characterised in that it is obtained from a filamentous epithelial product selected from among: fur, bristles, wool, hair, particularly human hair, or bird feathers, preferentially from among porcine bristles or human hair.

3. A method of obtaining a carrier protein preparation, characterised in that proteins susceptible to enzymatic digestion are removed from the filamentous products of vertebrate epithelia z in stages, wherein following the consecutive stages of pulverisation it is activated at least once with inorganic factors, treated with a proteolytic enzyme and washed, and the carrier protein preparation obtained in this fashion is isolated.

4. A method according to Claim 3, characterised in that as the filamentous product of vertebrate epithelia, one of the following sources is selected: fur, bristles, wool, hair, particularly human hair, or bird feathers, preferentially from among porcine bristles or human hair.

5. A method according to Claim 3, characterised in that the activation of intermediate preparations is performed using potassium or sodium hydroxide at a concentration of 0.1% to 4% by mass.

6. A method according to Claim 3, characterised in that digestion with a proteolytic enzyme is performed using an enzymatic preparation containing an enzyme selected from among: pepsin, chymotrypsin, trypsin, pancreatin or any combination of the above.

7. A method according to Claim 3, characterised in that the isolated carrier protein preparation loses less than 0.5% of its dry mass when further enzymatic hydrolysis is attempted.

8. An application of the carrier protein preparation according to Claims 1 and 2, or ones derived via a method according to Claims 3-7 in the manufacture of a carrier for stem cells.

9. An application according to Claim 8, characterised in that the carrier produced is used in a stem cell culture as a skeletal substance in the formation of conglomerates of stem cells destined for application in the regeneration of cells and tissues in humans.

10. An application according to Claim 9, characterised in that conglomerates of cells derived from stem cells cultured on the proteinaceous skeleton material obtained are used as implants for rescuing the function of damages neural, muscular and endocrine tissues.

11. An application according to Claim 10, characterised in that a patient's own hair is used to produce the carrier for cell agglomerate implants.

12. An application according to Claim 8, characterised in that the carrier produced is used in a stem cell culture devoid of animal sera and other culture materials of animal origin.

13. A cell conglomerate, characterised in that it contains cells and a carrier protein preparation according to one of Claims 1 to 6.

14. A conglomerate according to Claim 13, characterised in that stem cells or their derivatives are used.