Rotational dual chamber bioreactor: methods and uses thereof

Abstract

A rotational dual chamber bioreactor for cell culture in bi- and multi-layered scaffolds, in the context of tissue engineering (TE) and regenerative medicine strategies is disclosed, which comprises at least two dual culture chambers, a multiposition magnetic stirrer plate, and at least two flow pumps coupled to a monitor device to register biochemical parameters such as pH, pO.sub.2, glucose and urea sensors and physical parameters like pressure. A central barrier, with a hole to insert the bilayer scaffold, is used to culture cells with different conditions in each compartment of the chamber, avoiding medium exchange. Each chamber can rotate 180 (horizontal), which is promoted by magnetic stirrers, the multiposition stirrer plate is adapted dimensionally to the culture plate dimensions and also allows 180 vertical rotation. Each dual chamber has detachable caps for the top and the bottom chambers, the top cap to compress the scaffold thereby providing compressive stimulus with torsion simultaneously.

Claims

1. A bioreactor comprising: a multiposition magnetic stirrer plate; at least two dual chambers removably fixed to the multiposition magnetic stirrer plate by magnetic attraction via at least one magnet, the dual chambers comprising a top chamber and a bottom chamber, wherein the top chamber is a bi-compartmentalized culture chamber and comprises a top detachable cap, and wherein the bottom chamber is a detachable base housing the at least one magnet, avoiding direct contact with the culture medium; at least two independent flow pumps; and a platform for housing the multiposition magnetic stirrer plate, the at least two dual chambers and the at least two independent flow pumps, wherein the multiposition magnetic stirrer plate comprises a servo motor to vertically rotate the multiposition magnetic stirrer plate with the dual chambers, and at least two servo motor corresponding to each of the at least two dual chambers to independently horizontally rotate each dual chamber, the at least two servo motors placed between the dual chambers and the multiposition magnetic stirrer plate, and wherein the at least one magnet removably fixes by magnetic attraction the at least two dual chambers to the multiposition magnetic stirrer plate.

2. The bioreactor according to claim 1, wherein the horizontal rotational movement ranges from 0 to 180.

3. The bioreactor according to claim 2, wherein the rotational movement comprises two or more programmable speed scales ranging from zero to 0.12 sec/degrees.

4. The bioreactor according to claim 1, wherein the vertical rotational movement is applied to all dual chambers simultaneously.

5. The bioreactor according to claim 1, wherein the vertical rotational movement ranges from 0 to 180.

6. The bioreactor according to claim 1, wherein the dual chamber further comprises: a central barrier with a hole in the bi-compartmentalized culture chamber wherein the multilayered scaffolds are inserted; independent culture medium entries and outputs; compressible and/or detachable top caps; and microsensors to register biochemical and/or physical parameters.

7. The bioreactor according to claim 6, wherein the dual chamber is a dual culture chamber whose dimension is adapted to the culture plate dimension, preferably 38.4 mm in diameter and 17.5 mm in height.

8. The bioreactor according to claim 1, further comprising a top plate comprising adapters configured to enter into the top detachable cap of the dual chambers and supports attached to the platform.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Without intent to limit the disclosure herein, this application presents attached drawings of illustrated embodiments for an easier understanding.

(2) FIG. 1. Perspective view of the dual chamber (with open view for the inside), wherein a) is top cap of each dual chamber, b) represents the central part of the dual chamber where is comprised the bi-compartmentalization with the central hole for structure insertion and c) represents the bottom part adapted for insertion of a magnetic bar to fix the dual chamber to the stirred plate by magnetic attraction.

(3) FIG. 2. Cross section of the dual chamber.

(4) FIG. 3. Lateral view of the holder for the bioreactor, constituted by d) the screw of the support for fixing the stirred plate and e) fitting of the stirred plate.

(5) FIG. 4. Perspective view of the holder for the bioreactor

(6) FIG. 5. Perspective view (from bottom) of the top plate to be used for allowing mechanical compression. The plate is comprises the following features: f) adapters to entry in the top caps of dual chambers and g) supports that are attached by elastic to the dynamic platform.

(7) FIG. 6. Dynamic platform of the bioreactor that performs the rotating movements (horizontal and vertical). This platform is attached to and suspended by the holder.

(8) FIG. 7. Bilayered structures of Gellan gum-Gellan Gum/Hydroxyapatite; on the right: hydrated and stained with Alizarin red. In Alizarin red stained structure, the bottom part was stained with red (opaque in image) because of the presence of Hydroxyapatite particles. The top and brighter part is related with Gellan gum transparent part.

(9) FIG. 8. Bilayered structures profile for Hydroxyapatite % distribution and porosity. Left part of each structure is related with Gellan gum part, and right part with Gellan gum with Hydroxiapatite particles.

(10) FIG. 9. Rabbit Adipose derived stem cells from Fat Pad cultured on bilayered structures with gelatin in composition. DAPI/phalloidin and live/dead staining's for cells at the time-points of 24 h and 21 days of culturing. On left, it is possible to observe the cells stained with Phalloidin (white parts in the image). On right, it is possible to observe the living healthy cells (white colour).

DESCRIPTION OF PREFERRED EMBODIMENTS

(11) This invention refers to a rotational dual chamber bioreactor that is composed by a set of dual culture chambers, a multiposition magnetic stirrer plate, and flow pump(s).

(12) The dual culture chambers present two independent culture medium entries and outputs, which permit induction of independent and different shear flows, and a central separator with a hole for insertion of the scaffold, whereby there is integration of the two chambers. The dual chambers have a magnetic bar attached to the bottom part.

(13) The multiposition magnetic stirrer plate with 12 positions adapted for two 6 well tissue culture plates, which control independent horizontal movement for each position and vertical movement for all the plate.

(14) Rotational Dual Chamber Bioreactor:

(15) The dual chamber bioreactor is composed by a stirrer plate, which can be rotated vertically until 180. In addition, the stirrer plate have 12 positions where can be placed, by magnetic attraction, 12 dual chambers. Each one of the 12 positions can be independently controlled to rotate until 180 with 10 different speeds ranging from zero to 0.12 sec/degrees at no load.

(16) Dual Culture Chambers:

(17) The bioreactor can incorporate 12 dual culture chambers. The dual culture chambers have a central barrier with a hole to insert the bilayer scaffold. This kind of well serve to culture cells with different conditions in each chamber. The design of the dual culture chambers allows avoiding the mixture of the culture media. In the bottom part of the dual culture chamber is attached a magnetic bar to be attracted with the rotating position in the stirrer plate. Each chamber has detachable caps for the top and the bottom chambers. The top cap presents the possibly to compress the scaffold, allowing to test a compressive stimulus. The dual chambers have dimensions to adapt to commercial 6-well tissue culture plates (38.4 mm diameter, 17.5 mm height). This way, and being the top and bottom of the chambers detachable, the culture can be observed by microscopy. Microsensors can be added to the system to monitor several biochemical parameters, as oxygen tension, pH, temperature, or glucose and urea concentration and physical parameters like pressure. All of the pieces that compose the dual chambers are autoclavable.

(18) Stirrer Plate:

(19) The 12 multiposition stirrer plate is adapted dimensionally to two standard 6 well tissue culture plates. In each of the 12 positions can be inserted one of the dual culture chambers by magnetic attraction. In each position can rotate until 180 (horizontal), being the rotations per minute controlled independently. The rotation is promoted by magnetic stirring, having magnets performing the attraction between the bottom part of the well and the stirring position in the plate. Vertical 180 movement can also be applied for all the chambers together.

(20) The stirrer plate is controlled by a keyboard linked to a LCD display. The system is coordinated by an arduino (Atmel) synchronized with a servo control module. The stirrer plate can also incorporate a wi-fi system to control the stirring at distance, using computer software.

(21) All of the system can be placed inside an incubator.

(22) Osteochondral Tissue Development Using the Dual Chamber Rotational Bioreactor

(23) To develop an OC analogue, which can be further use in as an in vitro 3D tissue model, undifferentiated adipose derived stem cells (ASCs) isolated from Fat Pad are cultured in a bilayered scaffold, aiming at in situ cell differentiation into chondrocyte and osteoblast-like cells. The bilayered scaffolds comprise the cartilage- and bone-like layers, which are composed of gellan gum (GG) and GG with dispersed hydroxyapatite (HAp) particles, respectively.

(24) In vitro mature and homogeneous OC tissue formation is achieved by culturing ASCs within the GG-HAp/GG bilayered scaffold by means of using the dual chamber rotational bioreactor. An optimized chemical mediation is provided at each compartment of the dual chamber, i.e. in one compartment is provided the osteogenic medium and in the second compartment is provided the chondrogenic medium. Chondrogenic differentiation culturing cocktail is performed based on well-established protocols. Although osteogenic differentiation of ASCs can require additional Growth Factors, due to the presence of hydroxyapatite in the bone-like part of the scaffold, the conditions to maintain both cell types in co-culture needed to be optimized. The optimization, taking advantage of the dynamic culture system, accounts for the presence and the influence of osteogenic/chondrogenic mediators such as dexamethasone, L-ascorbic acid-2-phosphate, -glycerophosphate, BMPs, FGF, platelet-derived GF and TGF-.

(25) The mixture of the different culture medium is prevented due to the independent flow through the two compartments of the chamber. This maximizes the differentiation potential of the ASCs towards each lineage in the respective scaffold layers. The system characterized by the dual chamber rotational bioreactor and the produced living tissue aims to be used as a 3D in vitro OC model. These 3D tissue models make possible the continuous analysis of the growth factors production and allow culture conditions optimization, thus holding a great promise for application in tissue engineering and regenerative medicine, and screening of bioactive molecules or drugs (FIGS. 7, 8 e 9).

(26) The present disclosure is of course not in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof without departing from the basic idea of the invention as defined in the appended claims.

(27) The following claims set out particular embodiments of the invention.