RO132135A0 - Biomaterial based on collagen and expired human platelet lysate, to be used as scaffold for cell cultures and tissue engineering with biomedical applications - Google Patents

Abstract

The invention relates to a biomaterial to be used as a scaffold for cell cultures and tissue engineering with biomedical applications. According to the invention, the biomaterial is prepared by mixing 100 parts of collagen I or III solution prepared by a usual enzymatic treatment from bovine tendons and skin, having a concentration of 0.5...1%, an average molecular mass of 400000...500000, pH of 5.5...7.5, with 10 parts of platelet extract derived from expired platelet units, while completely homogenizing them for 2 h, and depositing the resulting biomaterial membranes on plane surfaces, to dry for 48 h, at a temperature of 37°C.

Description

COLOMEN-BASED BIOMATERIAL LIZED IN EXPIRED HUMAN PLATE UNITS FOR USE AS SCAFFOLD FOR CELLULAR CULTURES IN TISULAR ENGINEERING WITH BIOMEDICAL APPLICATIONS

The invention relates to a film-forming composite biomaterial, usable as a scaffold for cell cultures and tissue engineering of cartilage, with the effect of improving cell culture techniques.

Regenerative medicine / tissue engineering is a fast-growing multidisciplinary field that involves the life sciences, physics and engineering, with the goal of developing cells, tissues, and replacements of functional organs to repair, replace, or enhance the biological function that has been lost from the cause of congenital anomalies, injury, illness, or aging.

A tissue engineered product is currently defined as a product that contains or consists of cells or tissues and is used or administered to humans for the purpose of regenerating, repairing or replacing human tissue. The bioproduct from tissue engineering may contain cells, tissues of human or animal origin, or both. It may also contain additional substances, such as cellular products, biomolecules, biomaterials, chemicals and scaffolds, or matrices that provide physical support for cells / tissues.

The main components of cellular media include amino acids, organic and inorganic salts, vitamins, minerals, sugars, lipids and nucleic acids, their types and quantities varying according to the specific requirements of a particular cell type. Generally, the culture media used consisted of a basic medium (DMEM, MEM. RPMI and

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14/03/2017 others), supplemented with serum of animal origin, for example bovine fetal serum (SFB) and a mixture of antibiotics.

Current alternatives to FBS are based on components derived from human blood: plasma derivatives, serum, umbilical cord blood (UCB) serum and platelets, such as platelet lysate (HPL).

Although human platelet lysates (HPL) are being developed as a possible alternative to replacing SFB in MSC or other cell cultures, great caution is recommended when handling products of human origin, in order to prevent the transmission of infectious diseases. Therefore, it is preferred that human platelet lysates (HPLs) applied under clinical conditions are manufactured in accordance with the same strict regulations applied in the blood processing centers and in the hospital environment.

Another consideration involves the use of human donors required for the production of HPL and platelet rich plasma. But, to date, there is already a shortage of blood donors and platelets and therefore it is desirable not to compete with this need for fresh platelets with blood banks or transfusion centers.

It is also known that the life time of platelet units is very short, due to the increased risk of contamination with pathogens after several days of storage at room temperature, and which is not related to the functionality of platelets. The demand for platelets varies, which makes it difficult to estimate the need for platelet units for blood banks. It has been reported that about 20% of all platelet units expire before they can be used, and are therefore eliminated. After a short period of 5-7 days, platelet units are no longer considered safe for human transfusions, even though platelet function may still be intact.

As shown in OSIM Application no. A0050 dated 19.07.2016, the human platelet lysate (HPL or hPL) obtained from expired platelet units may be used as an alternative to SFB, similar to HPL produced from freshly isolated platelet units. The use of platelet lysate derived from the expiratory platelet units, axes = -fbPL.-e) can be made both as a substitute for SFB and as a supplement for increases in the growth of mechanics.

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14/03/2017 complete (with 10% SFB), at different concentrations (ranging from 2.5 - 10%), thus improving the most important parameters of the cell cultures used,

The problem solved by the present invention is obtaining a biocomposite biomaterial with biomedical applications in regenerative medicine and tissue engineering of cartilage, in the form of biocompatible membrane supplemented with platelet lysate from expired platelet units, with the role of improving the parameters of the cultures.

According to the invention, the biomaterial consists of 100 parts collagen solution (type

I) or (type III) obtained from tendons and bovine skin by the enzymatic treatment known and used in the art, with a concentration of 0.5-1% and an average molecular weight 400 000 500 000 containing 10-15 parts platelet extract derived from the units expired platelet (hPL-e).

The advantages of the invention consist of the following:

- The use of expired platelet units from the Transfusion Centers and the Hematology Institutes and in conclusion, a higher valorisation of this product;

- Obtaining HPL-e from platelets reached the limit of shelf life in transfusion centers as well as the possibility of storing them at -80 ° C, ensures the fulfillment of strict safety criteria, manufacture according to the accepted standards that apply to the production of transfusion materials for human patients, sterility and stability of storage conditions as well as complete traceability of the product from donation to use by the patient;

- Obtaining a much higher cellular expansion on collagen biomaterial containing HPL-e, compared to cell multiplication in the presence of SVF;

- total replacement of fetal bovine serum in cell cultures intended for tissue engineering.

Following is an example of embodiment of the invention entitled "Collagen-based and lysed biomaterial from expired human platelet units usable as a scaffold for cell culture and tissue engineering with biomedical applications.

A platelet concentrate (CP) transfusion unit, obtained by apheresis, with a volume of 50 ml / unit, containing 5.5x10 ¹⁰ in plasma, pH 6 at

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14/03/2017 room temperature (20 ° C), for 7 days after collection, under stirring, until expiration, and then stored at -80 ° C. Prior to processing, the frozen unit was thawed at 37 ° C on a water bath, which led to platelet lysis. The sample was centrifuged for 30 minutes at 4500g, the supernatant was recovered. To obtain a filtered lysate, the supernatant was filtered through a 0.45 µm filter (Millipore, MA, USA). The obtained filtrate was aliquoted into cryosubs and stored at -80 ° C. Before use, the aliquoted samples were after re-thawing 15 minutes at 4500g and only the supernatants were used.

Prepare a solution of collagen (type I) or (type III) obtained from tendons and bovine skin by the known enzymatic treatment used in the art, with a concentration of 0.5-1% and an average molecular weight 400 000 - 500 000, pH 5.5 to 7.5. In a planetary laboratory mixer, mix 100 parts 0.5 - 1% collagen solution and add 10 ml platelet extract. Stir for 2 hours. After complete homogenization, reduce the speed of the appliance and continue stirring until complete degassing, place on flat surfaces of polyethylene or Teflon and dry for 48 hours at 37 ° C. The resulting membranes are packed in polyethylene bags and sterilized with UV.

testing

The human chondrocytes (HCH Human Chondrocytes) provided by PromoCell at passage 2 were cultured on expired HPL collagen substrate, in 24-well plates (100 µl / well, 30 min without lid under hood and 24 h at 37 ° C). .

Chondrocytes were dispersed 4.5x10 ⁴ cells / well for each biomaterial sample and grown independently, and analyzed at 3 and 5 days of culture (Figure 1). For the respective cultures, a simple DMEM medium was used for future expansion and they were grown in an incubator with 5% CO2, 95% humidity and 37 ° C.

following the count of chondrocytes after 5 days of culture were found:

2.8x10 ⁵ cells / ml in the control sample with 10% SVF, (92% viability);

2.2x10® cells / ml in 10% HPL sample (93% viability).

The researches clearly indicated the possibility of cultivating chondrocytes on collagen scaffold having 10% HPL-e to improve cell culture techniques in tissue engineering of cartilage, with biorpeţijpâteh applications 1

Claims (1)

1. Collagen scaffold supporting membrane containing 10% HPL (platelet lysate derived from expired platelet units (hPL-e) to improve cell culture techniques in tissue engineering of cartilage, with biomedical applications.