Patent Publication Number: US-2021163882-A1

Title: System and method for seeding and culturing

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Application No. 62/483,031, filed Apr. 7, 2017, which is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present disclosure relates generally to tissue bioengineering and, more particularly, to methods and systems for seeding and culturing porous scaffold for tissue engineering of bone, cartilage, ligament, muscle, fat, teeth and tendons, among other possibilities. 
     BACKGROUND 
     Bioreactor cultivation of human stem cells in an osteogenic scaffold has been shown to support cell survival, differentiation, maturation and deposition of bone matrix, while restricting the development of unwanted lineages and facilitating a continued remodeling and vascularization following transplantation. 
     However, a traditional method of cultivating the scaffold often involves operations manually performed by a laboratory technician using multiple tools or devices, including soaking the scaffold in a media in a Petri dish, drying the scaffold, placing cells onto the scaffold and soaking the scaffold in a media bath to encourage cell growth. As such, there is a need for a cost-efficient and simplified mechanism to handle the cultivation of the scaffold without intense labor. The disclosed technology described herein addresses this need. In addition, the system described herein enables robust process control and maintenance of sterility by reducing user interaction in the process, SUMMARY 
     The disclosed technology relates to a system for seeding therapeutic active ingredients (TAIs) onto a porous scaffold. The system includes a first chamber for accommodating the scaffold, a TAI storage device for storing TAIs, at least one second chamber for storing TAI media, and a gas inlet for receiving gas from a compressed source. A flow circuit is coupled to (i) the first chamber, (ii) the TAI storage device, (iii) the second chamber and (iv) the gas inlet. The flow circuit delivers the TAIs, the TAI media and the gas to the scaffold. A pump pumps at least one of the TAIs, the TAI media and the gas in the flow circuit. The system also includes a plurality of valves for regulating the delivery of the TAIs, the TAI media and the gas to the scaffold. 
     In some embodiments, the flow circuit includes a media supply line and a media return line between the first chamber and the second chamber, a TAI supply line connected to the TAI storage device for delivering the TAIs, and a gas supply line connected to the gas inlet for delivering the gas. 
     In some embodiments, the plurality of valves include a media valve for regulating the delivery of the TAI media from the second chamber to the scaffold, a gas valve for regulating the delivery of the gas from the gas inlet, a TAI valve for regulating the delivery of the TAIs from the TAI storage device, a first chamber valve for regulating the delivery of the gas to the first chamber, and a waste valve for regulating a flow from the first chamber. 
     In some embodiments, the system includes a processor for regulating seeding and culturing of the scaffold by switching one or more valves between open and closed positions. The processor is configured to adjust a pump speed by the pump. 
     In some embodiments, the processor regulates treating the scaffold with the TAI media by operating the valves to control flow of the TAI media and the TAIs. 
     In some embodiments, the processor regulates removing an excessive amount of the TAI media from the scaffold with gas by adjusting the valves such that the gas valve, the first chamber valve and the waste valve are in an open position while remaining valves are in a closed position. 
     In some embodiments, the processor regulates pulsing pressurized gas bursts through the scaffold by repeatedly alternating each of the gas valve, the first valve and the waste valve between an open position and a closed position while remaining valves are in a closed position. 
     In some embodiments, the processor switches the gas valve to an open position and the first chamber valve to a closed position to generate pressurized gas prior to releasing a pressurized gas burst. 
     In some embodiments, the processor switches the gas valve to a closed position and the first chamber to an open position to release the pressurized gas burst into the first chamber. 
     In some embodiments, the processor regulates releasing the TAIs from the TAI storage device by adjusting the valves such that the TAI valve and the waste valve are in an open position while remaining valves are in a closed position. 
     In some embodiments, the processor regulates pumping the TAIs into the first chamber by adjusting the valves such that the media valve, the first chamber valve and the waste valve are in an open position while remaining valves are in a closed position. 
     In some embodiments, the system includes a scaffold turner, driven by a motor (which include various types of motors such as rotary actuator, stepper, brushed, brushless, pneumatic, hydraulic, etc.), for rotating the scaffold accommodated in the first chamber through a plurality of positions. 
     In some embodiments, the processor regulates TAI adhesion on the scaffold by instructing the motor that drives the scaffold turner to rotate the scaffold through the plurality of positions. 
     In some embodiments, the system includes a motor for driving the pump. 
     In some embodiments, the processor regulates TAI culturing by instructing the motor that drives the pump to pump the TAI media into the first chamber for a predetermined period of time. 
     In some embodiments, the graft includes one or more of the following tissues: bone, cartilage, ligament, muscle, fat, skin, nerves, lymph nodes, bone marrow, teeth and tendons. In certain embodiments, the graft is a bone graft. 
     In some embodiments, the compressed source provides CO 2 , N 2  or mixed gases. 
     Another aspect of the disclosed technology relates to a method for seeding and culturing TAIs into a porous scaffold. The method includes accommodating a scaffold in a first chamber. A flow circuit delivers TAIs, TAI media and gas to the scaffold from a TAI storage device, at least one second chamber and a gas inlet. A pump pumps at least one of the TAIs, the TAI media and the gas in the flow circuit. A processor regulates the delivery of the TAIs, the TAI media and the gas to the scaffold via a plurality of valves. 
     In some embodiments, the plurality of valves include a media valve for regulating the delivery of the TAI media from the second chamber, a gas valve for regulating the delivery of the gas from the gas inlet, a TAI valve for regulating the delivery of the TAIs from the TAI storage device, a first chamber valve for regulating the delivery of the gas to the first chamber, and a waste valve for regulating a flow from the first chamber. 
     In some embodiments, the processor regulates pulsing pressurized gas bursts through the scaffold by repeatedly alternating each of the gas valve, the first chamber valve and the waste valve between an open position and a closed position while remaining valves are in a closed position. 
     In some embodiments, the processor switches the gas valve to an open position and the first chamber valve to a closed position to generate pressurized gas prior to releasing a pressurized gas burst. 
     In some embodiments, the processor switches the gas valve to a closed position and the first chamber valve to an open position to release the pressurized gas burst into the first chamber. 
     In some embodiments, the graft includes one or more of the following tissues: bone, cartilage, ligament, muscle, fat, skin, nerves, lymph nodes, bone marrow, teeth and tendons. In certain embodiments, the graft is a bone graft. 
     A further aspect of the disclosed technology relates to a method of treating a surface of a porous scaffold. The method includes using liquid and gas pressure to wet and coat the surface of the porous scaffold with a thin layer to facilitate wicking of a cell solution, resulting in a uniform seeded scaffold. 
     In some embodiments, the method includes accommodating the porous scaffold in a first chamber; delivering TAIs, TAI media and gas to the scaffold via a flow circuit from a TAI storage device, at least one second chamber and a gas inlet; pumping at least one of the TAIs, the TAI media and the gas in the flow circuit by a pump; and regulating the delivery of the TAIs, the TAI media and the gas to the scaffold by a processor via a plurality of valves. 
     Another aspect of the disclosed technology relates to a preassembled kit for integration into an automated system for scaffold seeding and culturing. The preassembled kit includes a scaffold, a first chamber for accommodating the scaffold, a second chamber for storage purposes, and a flow circuit for connecting the bioreactor with the reservoir. The scaffold, the first chamber, the second chamber and the flow circuit are assembled to form an input to the automated system for scaffold seeding and culturing. The automated system after integration with the preassembled kit includes: the first chamber for accommodating the scaffold; the second chamber for storing TAI media; a therapeutic active ingredient (TAI) storage device for storing TAIs; a gas inlet for receiving gas from a compressed source; the flow circuit coupled to (i) the first chamber, (ii) the TAI storage device, (iii) the second chamber and (iv) the gas inlet, said flow circuit delivering the TAIs, the TAI media and the gas to the scaffold; a pump for pumping at least one of the TAIs, the TAI media and the gas in the flow circuit; and a plurality of valves for regulating the delivery of the TAIs, the TAI media and the gas to the scaffold. 
     Various aspects of the described example embodiments may be combined with aspects of certain other example embodiments to realize yet further embodiments. It is to be understood that one or more features of any one example may be combined with one or more features of the other example. In addition, any single feature or combination of features in any example or examples may constitute patentable subject matter. Other features of the technology will be apparent from consideration of the information contained in the following detailed description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following Detailed Description of the technology is better understood when read in conjunction with the appended drawings. For the purposes of illustration, there is shown in the drawings exemplary embodiments, but the subject matter is not limited to the specific elements and instrumentalities disclosed. Components in the figures are shown for illustration purposes only, and may not be drawn to scale. 
         FIG. 1  is a schematic diagram of an automated seeding and culturing system according to one embodiment of the present technology. 
         FIG. 2  is a schematic illustration of turning mechanism by a scaffold turner according to one embodiment of the present technology. 
         FIG. 3  is a schematic diagram of a fluid circuit of the automated seeding and culturing system according to one embodiment of the present technology. 
         FIG. 4  is a schematic diagram of an electrical circuit coupled to the fluid circuit of the automated seeding and culturing system according to one embodiment of the present technology. 
         FIG. 5  is a schematic illustration of a scaffold during (a) a pre-wetting process and (b) a TAI delivery and adhesion process according to one embodiment of the present technology. 
         FIG. 6  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to introduce a flow of media into a first chamber according to one embodiment of the present technology. 
         FIG. 7  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to introduce gas into the first chamber according to one embodiment of the present technology. 
         FIG. 8  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to introduce pressurized gas bursts into the first chamber according to one embodiment of the present technology. 
         FIG. 9  is a schematic illustration of a scaffold during a pre-wetting process according to one embodiment of the present technology. 
         FIG. 10  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to introduce a TAI solution to a point just before the first chamber according to one embodiment of the present technology. 
         FIG. 11  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to pump the TAI solution into the first chamber according to one embodiment of the present technology. 
         FIG. 12  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to rotate the scaffold through different positions according to one embodiment of the present technology. 
         FIG. 13  is a schematic diagram illustrating the scaffold at different rotation positions according to one embodiment of the present technology. 
         FIG. 14  is a schematic diagram comparing different effects as a result of rotating scaffold to different positions. 
         FIG. 15  is a schematic diagram of a circuit configuration of the automated seeding and culturing system to introduce continuous media flow into the first chamber during a culturing process according to one embodiment of the present technology. 
         FIG. 16  is a schematic diagram of an alternative circuit configuration of the automated seeding and culturing system according to one embodiment of the present technology. 
         FIG. 17  illustrates homogenous delivery and attachment of ATIs throughout a large porous scaffold. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent to those skilled in the art that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings. 
     1. Overview 
     The present technology relates to engineering a custom-designed autologous graft by cultivating human stem therapeutic active ingredients (TAIs) in an osteogenic scaffold. The TAIs may include one or a combination of the following: cells, drugs, proteins and hydrogel, among other possibilities. The graft may include one or more of the following tissues: bone, cartilage, ligament, muscle, fat, skin, nerves, lymph nodes, bone marrow, teeth and tendons, among other possibilities. A patient specific graft may be designed based on a precise three-dimensional (3D) model of an anatomical defect from a patient&#39;s computerized tomography (CT) scan. Using the 3D image, an anatomically-shaped scaffold may be fabricated. 
     The present technology relates a system  100  for cultivating the graft ready for implantation by seeding and culturing the scaffold.  FIG. 1  provides a schematic diagram of the system  100  according to one embodiment. The system  100  may be an all-in-one device that handles seeding and culturing of the scaffold. For instance, the system  100  may perform, but is not restricted to, all of the following, pre-wetting the scaffold including gas purging, impregnating the scaffold with TAIs, rotating the scaffold to provide uniform seeding, delivering TAI media to feed the TAIs, and removing wastes produced by the TAIs. 
     With reference to  FIG. 1 , the system  100  may include a first chamber  102  that provides an environment for growing a scaffold into a graft. The first chamber may include a mold to accommodate the scaffold. The mold may be custom-built based on the 3D image so as to conform to the configuration of the scaffold. 
     The system  100  may perfuse the scaffold with TAI s by delivering a TAI solution to the scaffold. The TAI solution may be stored in a TAI storage device  106 . The TAI storage device  106  may be a syringe. The TAI solution may include a concentrated suspension of TAIs. The TAIs may be derived or extracted from the patient&#39;s own tissue. The TAI solution may be delivered to the first chamber  102  so as to impregnate the scaffold with the TAIs. The TAIs may proliferate in the scaffold and expand into sufficient quantity so as to form a viable graft. 
     The system  100  may also include one or more second chambers  108  for storing food sources for the TAIs, such as TAI media, to be delivered to the first chamber  102  so as to feed the TAIs. The second chamber  108  may be a TAI media chamber  108 . The system  100  may include an gas inlet  110  for receiving gas from a compressed source, and the gas is to be delivered to the first chamber  102 . The compressed source may provide CO 2 , N 2  or mixed gases. 
     The first chamber  102  may include an inlet  102   a  for receiving the TAI media, TAIs, gas for seeding and culturing the scaffold stored therein. The first chamber may include an outlet  102   b  for releasing gas including gas bubbles, excessive TAI media, and wastes produced by the TAIs. 
     The TAI media chamber  108  may have an outlet  108   a , coupled to a media supply line  120 , for releasing the TAI media to feed TAIs in the first chamber  102 . The TAI media chamber  108  may have an inlet  108   b , coupled to a media return line  122  for receiving TAI media returned from the first chamber  102  as well as the wastes produced by the TAIs in the first chamber  102 . The TAI media chamber  108  may store the wastes produced by the TAIs. The TAI media chamber  108  may also receive and collect gas bubbles produced by the first chamber  102  via the media return line  122 . 
     The media supply line  120  may connect the outlet  108   a  of the TAI media chamber  108  to the inlet  102   a  of the first chamber  102 . The media supply line  120  may travel through a pump  112 , driven by a motor  114 , for regulating fluid movement in the media supply line  120 . The pump  112  may be a peristaltic pump, a syringe, or a diaphragm pump, among other possibilities. The motor  114  may be chosen from various types of motors such as rotary actuator, stepper, brushed, brushless, pneumatic and hydraulic motors, among other possibilities. Further, the media supply line  120  may travel through a media valve  124  and a first chamber valve  126 . Each valve may have an open position to allow a flow through the media supply line  120 , and a closed position to block the flow. For example, the media valve  124  may regulate the flow between the TAI media chamber  108  and the pump  112 , while the first chamber valve  126  may regulate the flow between the pump  112  and the first chamber  102 . The media valve  124  may regulate the delivery of the TAI media from the TAI media chamber  108  chamber to the scaffold. 
     The media return line  122  may connect the inlet  108   b  of the TAI media chamber  108  to the outlet  102   b  of the first chamber  102 . Further, the media return line  122  may travel through a waste valve  128 . The waste valve  128  may regulate a flow from the first chamber  102  to the TAI media chamber  108 . For example, the waste valve  128  may have an open position to allow the flow through the media return line  120 , and a closed position to block the flow. The media return line  122  may convey the TAI media from the first chamber  102  back into the TAI media chamber for reuse. 
     The TAI storage device  106  may be coupled to a TAI supply line  134 . The TAI supply line  134  may travel through a TAI valve  136 . The TAI valve  136  may regulate a flow of TAIs in the TAI supply line  134 . For example, the TAI valve  136  may have an open position to allow the flow of TAIs through the TAI supply line  134 , and a closed position to block the flow of TAIs. The TAI supply line  134  may meet the media supply line  120  at a coupling point  131   a , where the TAIs from the TAI supply line  134  flow into the media supply line  120 . 
     The gas inlet  110  may be a sterile gas filter such as a PTFE membrane or a high-efficiency particulate air (HEPA) filtered inlet. The gas inlet  110  may be temperature and atmosphere controlled. The gas inlet  110  may be coupled to a gas supply line  130 . The gas supply line  130  may travel through a gas valve  132 . The gas valve  132  may regulate the gas flow in the gas supply line  130 . For example, the gas valve  132  may have an open position to allow the gas flow through the gas supply line  130 , and a closed position to block the gas flow. The gas supply line  130  may meet the media supply line  120  at a coupling point  131   b , where the gas from the gas supply line  130  flows into the media supply line  120 . When the media supply line  120  carries the gas, the pump  112  and the first chamber valve  126  may charge and purge gas to and from the first chamber  102 . 
     The lines  120 ,  122 ,  130  and  134  described herein may be soft tubes such as platinum cured silicone tubes. The lines  120 ,  122 ,  130  and  134  may form a flow circuit. The valves  124 ,  126 ,  128 ,  132  and  136  described herein may be solenoid valves, such as pinch valves. The valves may control flow through the lines or through the flow circuit. In one embodiment, the pinch valves may squeeze the soft tubes so as to restrict flow through the soft tubes. 
     The system  100  may include a scaffold turner or rotary holder  116 . The scaffold turner  116  may serve as a clamp for holding the first chamber  102 . The scaffold turner  116  may have flexible arms  116   a  so as to hold first chambers of various configurations, such as various geometries. The scaffold turner  116 , driven by a motor  118 , may rotate the first chamber  102  so as to enable uniform scaffold seeding. The motor  118  may be chosen from various types of motors such as rotary actuator, stepper, brushed, brushless, pneumatic and hydraulic motors, among other possibilities  FIG. 2  provides a schematic illustration of the scaffold turner  116  in action which rotates the first chamber  102  as well as the scaffold  104  contained therein. 
       FIG. 3  is a schematic diagram of a fluid circuit of the system  100  according to one embodiment of the present technology. In  FIG. 3 , three separate inputs including the gas, TAI media and TAI solution are respectively introduced from the gas inlet  110 , the TAI media chamber  108 , and the TAI storage device  106  into the first chamber  102  that houses the scaffold. Each of these inputs may be isolated from the first chamber  102  by one or more dedicated valves. For example, the gas, TAI media and TAI solution may be isolated from the first chamber  102  by the gas valve  132 , the media valve  124 , and the TAIs valve  136 , respectively. The pump  112  and the first chamber valve  126  may charge and purge gas to and from the first chamber  102 . The waste valve  128  may regulate flow from the first chamber  102  to the TAI media chamber  108 . 
       FIG. 4  is a schematic diagram of an electrical circuit coupled to the fluid circuit of the system  100  according to one embodiment of the present technology. As illustrated in  FIG. 4 , operations of each valve  124 ,  126 ,  128 ,  132  and  136  may be controlled by a processor or microcontroller  140 . The processor  140  may include pre-loaded code, which may be uploaded or edited by a user via a user interface  148 . The processor  140  may receive geometric properties, such as void volumes, of the scaffold  104  and the first chamber  102 . Such information may be provided by the user via the user interface  148 . Based on the geometric properties, the processor  140  may determine time for switching each valve between its open and closed positions so as to control the flow, and instruct each valve to perform switching via a relay board  142 . 
     Further, the processor  140  may control the motor  114  that drives the pump  112 , and the motor  118  that drives the scaffold turner  116 . The processor may determine parameters for operating the motors, and communicate with the motors via their respective motor drivers  144 . The processor may adjust a pump speed by the pump  112 . 
     The system  100  may include one or more sensors or sampling ports  146  for taking chemical and physical readings or samples from the flow in the system  100 . The processor  104  may analyze the readings to determine if the system  100  has experienced any failure. The processor  104  may also rely on the readings to generate experimental data. 
     The user interface  148  may provide a screen which displays operation information of the system  100 . The user interface  148  may also provide switches for receiving user inputs. The user interface  148  may have a USB port for receiving algorithms to control the system  100 . 
     For simplicity purposes, a power supply is not illustrated in  FIG. 4 . 
     2. Seeding and Culturing Processes 
     The system  100  may regulate seeding and culturing of the scaffold according to embodiments described below. 
     2.1 Initialization 
     At initialization, the processor  140  may be set with constant parameters for operating the motors  114  and  118  in accordance with motor specifications. Pins for the processor  140  may also be initialized. At initialization, the processor  104  may also be set with seeding parameters, such as variables describing how long media is cycled, the first chamber rotation rate and positions, and pressurization time. Further, the processor  104  may be set with pump speeds and pump volumes for different functions. Motor positions and program locations may also be initialized. 
     At initialization, the processor  140  may switch all valves to their closed positions. The user interface  148  may display instructions to the user, such as the following: (1) ask the user to load the first chamber  102  and the lines  120 ,  122 ,  130  and  134 , (2) ask the user to unlock the media flow, (3) ask the user to enter the first chamber type. Based on the first chamber type, the processor may determine the volume of the first chamber and the flow rate for perfusing the TAI media. In one embodiment, the volume of the first chamber and the flow rate may be provided by the user. 
     2.2 Seeding 
     The seeding process may include (1) a pre-wetting process (phases 1-3 described below), (2) a TAI delivery process (phases 4-5 described below), and (3) a TAI adhesion process (phase 6 described below). 
     As shown in  FIG. 5 , during the pre-wetting process, TAI media is delivered into the scaffold and fills void spaces in the scaffold including its walls. As part of the pre-wetting process, gas is introduced into the scaffold and fills void spaces in the scaffold. Pre-wetting is important for TAI adhesion to a scaffold. During the TAI delivery process, TAIs are delivered to the scaffold. During the TAI adhesion process, TAIs may climb into crevices of the pre-wet walls of the scaffold. 
     The pre-wetting process may wet all inner surfaces of the scaffold. The pre-wetting process may provide benefits for the TAI delivery process and the TAI adhesion process. For example, as a result of the pre-wetting process, the TAI solution may be encouraged to climb into crevices of walls of the scaffold by capillary action and surface tension. Accordingly, the TAI solution may move into hard-to-reach places where the TAI solution otherwise would not travel to through its natural course. As a result, the pre-wetting process ultimately enables the TAI solution to cover and form adhesion with more surface areas of the scaffold. The pre-wetting process is particularly important for scaffolds that have porous structures, as the porous structures contain many roundish cavities with potentially small entrances and sharp features that hinder fluid flow. With the benefit of pre-wetting, inputted TAIs can readily climb into the crevices of the pre-wet walls. 
     Detailed discussion of each phase of the seeding process is provided below 
     2.2.1 Phase 1: Media Rinse (Pre-Wet Part 1) 
     Prior to the pre-wetting process, a completely dry scaffold may be placed in the first chamber  102 . The scaffold may have a 3D porous structure.  FIG. 6  illustrates a circuit configuration of the system  100  in the first phase of the pre-wetting process. The processor may regulate treating the scaffold with the TAI media by operating the valves to control flow of the TAI media and the TAIs. As shown in  FIG. 6 , the processor  140  may switch the media valve  124 , the first chamber valve  126 , and the waste valve  128  to their open positions, while the gas valve  132  and the TAI valve  136  are in their closed positions. A predetermined amount of the TAI media from the TAI media chamber  108  may be pumped into the first chamber  102  via the pump  112 . As the TAI media is pumped into the first chamber  102 , the TAI media may fill void spaces in the scaffold including its walls. As a result, the scaffold stored in the first chamber  102  may be rinsed and soaked in the forced flow of TAI media. The forced flow of the TAI media may increase the amount of the surface area of the scaffold wetted by the TAI media. For example, all surfaces of the scaffold may be wetted by the TAI media. 
     After the TAI media is delivered into the first chamber  102 , the processor  140  may switch the media valve  124 , the first chamber valve  126  and the waste valve  128  to their closed positions. 
     2.2.2 Phase 2: Gas Purge  1  (Pre-Wet Part 2) 
       FIG. 7  is a schematic diagram of a circuit configuration of the system  100  in the second phase of the pre-wetting process. In this phase, the processor  140  may first, as an optional step, initialize the first chamber position to prevent tube tangling. For example, the processor  140  may instruct the motor  118  to rotate the scaffold turner  116  so as to rotate the first chamber  102  and the scaffold contained therein to a particular position. For example, the first chamber  102  may be rotated to a position as shown in  FIG. 1 , where the inlet  102   a  is at the top and the outlet  102   b  is at the bottom. As a result of the rotation of the first chamber  102 , the scaffold contained therein may be rotated to position 3 (180 rotation) as illustrated in  FIG. 13 , and also illustrated in  FIG. 14 . As shown in  FIG. 14 , when the inlet  102   a  is at the top and the outlet  102   b  is at the bottom, gravity assists the flow in the first chamber, and helps fluid drainage. In other examples, the first chamber  102  and the scaffold contained therein may be rotated to other suitable positions. This rotation step may be optional, such that the processor  140  may omit the rotation step and start the second phase of the pre-wetting process with the next step. 
     Next, the processor  140  may open the gas valve  132 , the first chamber valve  126  and the waste valve  128 . Thereafter, gas from the gas inlet  110  may be pumped into the first chamber via the pump  112 . As gas is introduced through the scaffold, excessive TAI media in the void spaces of the scaffold may be removed from the scaffold, and may be returned back to the TAI media chamber  108 . As the bulk of the TAI media is removed from the scaffold, a thin layer of the TAI media may remain in inner and outer walls of the scaffold. After a brief period of time, the processor  140  may close the gas valve  132 , the first chamber valve  126  and the waste valve  128 . 
     The second phase may facilitate volumetric distribution of TAIs in later phases 
     2.2.3 Phase 3: Gas Purge  2  (Pre-Wet Part 3) 
       FIG. 8  is a schematic diagram of a circuit configuration of the system  100  in the third phase of the pre-wetting process. In this phase, the processor  140  may determine a number of gas bursts. 
     Each gas purge may include a gas charging step and a gas releasing step. At the gas charging step, the processor  140  may open the gas valve  132  and the waste valve  128  while other valves remain closed. Under the control of the pump  112 , gas may flow from the gas inlet  110  into the gas supply line  130 . Since the first chamber valve  126  is at its closed position, pressurized gas may be temporarily stored in the line or lines between the gas inlet  110  and the first chamber valve  126 . The line or lines that carry the pressurized gas may be made of a flexible material, and may inflate like a balloon when one end is held closed and gas is pushed into it. 
     At the gas releasing step, the processor  140  may first close the gas valve  132  prior to a gas burst. Then, the processor  140  may open the first chamber valve  126  to release one pulse of a pressurized gas burst into the first chamber  102 , while the waste valve  128  remains open. This gas burst may wet the scaffold in the first chamber  102  by strongly compressing the TAI media in the first chamber  102 . The gas burst may also eject any lingering TAI media in the void spaces of the scaffold. After a gas burst is released, the processor  140  may close the first chamber valve  126  and the waste valve  128  to prepare for the next gas burst. 
     The processor  140  may repeatedly alternate the gas charging and releasing steps until the determined number of gas bursts are released. Table I produced below provides an exemplary sequence of valve operations in this third phase 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Function 
                 Valve 132 
                 Valve 126 
                 Valve 128 
               
               
                   
                   
               
             
            
               
                   
                 Charging 
                 Open 
                 Closed 
                 Open 
               
               
                   
                 Pre-Burst 
                 Closed 
                 Closed 
                 Open 
               
               
                   
                 Burst 
                 Closed 
                 Open 
                 Open 
               
               
                   
                 Pre-Charge 
                 Closed 
                 Closed 
                 Closed 
               
               
                   
                   
               
            
           
         
       
     
       FIG. 9  is a schematic illustration of transformation of a scaffold through the three phases of the pre-wetting process. As illustrated in  FIG. 9 , step (a) represents a scaffold  104  in its completely dry state prior to the pre-wetting process. Step (b) represents the first phase of the pre-wetting process during which the TAI media is introduced into the scaffold  104 . A layer  202  of TAI media is formed on a wall of the scaffold  104 , along with excessive/residual TAI media  204 . Step (c) represents the second phase of the pre-wetting process in which excessive TAI media  204  is removed from void spaces by gas. As illustrated in step (c), some residual TAI media  204  may still cling to some inner surfaces of the scaffold  104 . Steps (d) and (e) represent the third phase of the pre-wetting process in which consecutive pulses of pressurized gas may remove residual TAI media off the walls of the scaffold  104 . 
     The method of using gas bursts to eject excessive TAI media and wet the scaffold may provide many advantages. The gas bursts may aid in the removal of fluid in the pore spaces especially the regions with smaller pores which may be more restricted, tortuous and harder to reach. After gas purge, the scaffold pores are free of fluid resulting in open space with wetted surface which may aid the delivery and distribution of the TAI solutions. Casting, painting, and surface coating industries may use sprays, which generally rely on centrifugal forces and gravity, to evenly coat surfaces. Using gas bursts would be inefficient in those industries. 
     2.2.4 Phase 4: TAI Delivery  1   
     Subsequent to the pre-wetting process, the TAI solution may be introduced into the scaffold through a TAI delivery process. The TAI delivery process may include two phases.  FIG. 10  is a schematic diagram of a circuit configuration of the system  100  in the first phase of the TAI delivery process, which also corresponds to phase 4 of the overall seeding and culturing process. In this phase, the processor  140  may, as an optional step, rotate the scaffold turner  116  so as to rotate the first chamber  102  and the scaffold contained therein to a different position. For example, the first chamber  102  may be rotated to a position such that the outlet  102   b  is at the top, while the inlet  102   a  is at the bottom. As a result, the scaffold contained therein may be placed to position 1 as illustrated in  FIG. 13 , and also illustrated in  FIG. 14 . Such rotation may encourage capillary action, preventing the media from flowing in a single downward direction under gravity. For instance, as shown in  FIG. 14 , when the outlet  102   b  is at the top, and the inlet  102   a  is at the bottom, the TAI media flows through the scaffold&#39;s pores under the influence of capillary action, opposing the force of gravity. In other examples, the first chamber  102  and the scaffold contained therein may be rotated to other suitable positions. This rotation step may be optional, such that the processor  140  may omit the rotation step and begin the TAI delivery process with the next step. 
     At the next step, the processor  140  may open the TAI valve  136  and the waste valve  128 . Thereafter, the processor  140  may instruct the user interface  148  to display instructions to the user to inject the TAI solution containing the TAIs from the TAI storage device  106 . In one embodiment, instead of manual injection, the TAI solution may be injected via a pump. The TAI solution may flow through the TAI supply line  134 , and stop prior to the first chamber  102 . 
     2.2.5 Phase 5: TAI Delivery  2   
       FIG. 11  is a schematic diagram of a circuit configuration of the system  100  in the second phase of the TAI delivery process, which also corresponds to phase 5 of the overall seeding and culturing process. In this phase, the processor  140  first closes the TAI valve  136 , and then opens the medial valve  124  and the first chamber valve  126 . Next, the processor  140  may instruct the motor  114  to activate the pump  112 . Under the control of the pump  112 , the TAI media may flow into the media supply line  120  from the TAI media chamber  108 , and push the TAI solution into the first chamber  102 . As a result, a concentrated TAI solution may be placed on the scaffold and absorbed into the scaffold. Thereafter, the processor  140  may close the waste valve  128 . 
     During this phase, the TAI solution may be pumped forward and carefully positioned to fill the scaffold volume. This is important for the efficiency of TAI seeding. This phase ensures minimal TAIs are wasted in a location that is not the scaffold. 
     2.2.6 Phase 6: TAI Adhesion 
     Once the TAIs are delivered into the first chamber  102 , the TAIs need some time to adhere to the scaffold. This process of placing TAI solution and resting may be done multiple times, and may coordinate with changing the scaffold&#39;s orientation.  FIG. 12  is a schematic diagram of a circuit configuration of the system  100  for rotating the scaffold through different positions. In this phase, the waste valve  128  remains closed, and the first chamber  102  stays in a closed condition. The processor  140  may instruct the motor  118  to rotate the scaffold turner  116  so as to periodically rotate the scaffold through various positions. By rotating the scaffold through various positions, TAIs may be distributed over different areas of the scaffold to compensate for gravitational bias. For examples, TAIs that are not attached to any area of the scaffold may fall to other areas of the scaffold that do not yet have TAIs. The scaffold turner  116  may repeat the rotation multiple times. In one embodiment, the scaffold turner  116  may periodically move back and forth in 180 degrees, and stay static for a period of time between motions to let the TAIs settle or allow the TAIs to form attachments with the scaffold. 
       FIG. 13  is a schematic diagram illustrating exemplary positions of the scaffold according to one embodiment. For instance, the scaffold may be rotated or turned through five distinct positions, including a 0-degree rotation corresponding to position 1, a 90-degree rotation corresponding to position 2, a 180-degree rotation corresponding to position 3, a 270-degree rotation corresponding to position 4 and a 360-rotation corresponding to position 5. As illustrated in  FIG. 13 , with the rotation or turning mechanism, uniform distribution and attachment of the TAIs over the scaffold is achieved, avoiding irregular distribution of the TAIs which otherwise would occur in the absence of the rotation or turning mechanism. The degree of rotation may be defined with respect to the bioreactor  102 . For example, as shown in  FIG. 14 , the 180-degree rotation may correspond to a position where the inlet  102   a  is at the top, while the outlet  102   b  is at the bottom. The 0-degree rotation may correspond to a position where the inlet  102   a  is at the bottom, while the outlet  102   b  is at the top. 
     In one embodiment, while the waste valve  128  remains closed, the first chamber  102  may be pressurized periodically by pumping the TAI media against the closed waste valve  128 . 
     The above phases (phase 1 to phase 6) complete the seeding process. 
     2.3 Phase 7: TAI Culturing 
     As a result of the TAI seeding process, the TAIs may form sufficient attachments with the scaffold. As nutrients are consumed by the TAIs, wastes are also produced by the TAIs. The TAI media inside the first chamber  102  thus needs to be refreshed from time to time. For that reason, the system  100  may enter a TAI culturing phase to refresh the TAI media periodically and also to continue the perfusion period. The TAI culturing phase may last for a period of weeks. 
       FIG. 15  is a schematic diagram of a circuit configuration of the system  100  in the TAI culturing phase. In this phase, the processor  140  may repeatedly refresh the TAI media from time to time. The processor  140  may perform the following steps to refresh the TAI media each time. 
     The processor  140  may first close all valves. Next, the processor  140  may instruct the motor  118  to rotate the scaffold turner  116  so as to rotate the scaffold to a particular position. For example, the scaffold may be placed to position 3 as illustrated in  FIG. 13 , or other suitable positions. Thereafter, the processor  140  may open the waste valve  128 , the first chamber valve  126  and the media valve  124 . Next, the processor  140  may activate the motor  114  for the pump, and pump the TAI media from the TAI media chamber  108  into the first chamber  102  for a predetermined period of time. During this predetermined period of time, the TAI media may be changed. 
     Once the predetermined period of time lapses, the processor  140  may perform one or more of the following: (1) reverse the media flow to perfuse the scaffold with different flow directions, (2) instruct the motor  118  to rotate the scaffold turner  116  so as to rotate the scaffold to another position, e.g., position 0 as illustrated in  FIG. 13 , or other suitable positions, (3) stop pumping, and (4) close all valves. By rotating the scaffold to various positions, media may flow to various locations within the scaffold to encourage uniform TAI growth, avoiding TAI growth in one concentrated location that may otherwise occur in the absence of rotation. 
     3 Alternative Embodiments 
       FIG. 16  is a schematic diagram of an alternative circuit configuration of the system  100 . In this configuration, the waste valve  128  may be optional. The removal of the waste valve  128  may limit the performance of the system  100  at the TAI adhesion phase (Phase 6). 
     In another alternative embodiment, the user may engage in a manual process of rotating the scaffold turner  116  or operating the pump  112 . For instance, the user may select such manual operation through the user interface  148 . Once the user selects the manual operation, the processor  140  may open the media valve  124 , the first chamber valve  126  and the waste valve  128 . The processor  140  may instruct the user interface  148  to display a message asking the user for a pump rate. The user may use switches to rotate the scaffold turner  116 , or pump TAI media at user defined speeds. The user may pause the process at any time by selecting a pause option at the user interface. 
     In some embodiments, a surface of a porous scaffold may be treated using liquid and gas pressure to wet and coat the surface of the porous scaffold with a thin layer to facilitate wicking of a cell solution, resulting in a uniform seeded scaffold. The scaffold may be seed and cultured according to methods described earlier. 
     In some embodiments, the scaffold, the first chamber, the second chamber and the flow circuit may form a preassembled kit for integration into an automated system for scaffold seeding and culturing. The scaffold, the first chamber, the second chamber and the flow circuit may be assembled to form an input to the automated system for scaffold seeding and culturing. The automated system after integration with the preassembled kit may perform seeding TAIs onto the scaffold according to methods described earlier. 
       FIG. 17  illustrates homogenous delivery and attachment of fluorescent labeled ATIs  1702  throughout a large porous scaffold  1704  which for example may have a diameter greater than 5 cm. 
     While certain implementations of the disclosed technology have been described in connection with what is presently considered to be the most practical and various implementations, it is to be understood that the disclosed technology is not to be limited to the disclosed implementations, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. 
     Certain implementations of the disclosed technology are described above with reference to block and flow diagrams of systems and methods and/or computer program products according to example implementations of the disclosed technology. It will be understood that one or more blocks of the block diagrams and flow diagrams, and combinations of blocks in the block diagrams and flow diagrams, respectively, can be implemented by computer-executable program instructions. Likewise, some blocks of the block diagrams and flow diagrams may not necessarily need to be performed in the order presented, or may not necessarily need to be performed at all, according to some implementations of the disclosed technology. 
     These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means that implement one or more functions specified in the flow diagram block or blocks. 
     Implementations of the disclosed technology may provide for a computer program product, comprising a computer-usable medium having a computer-readable program code or program instructions embodied therein, said computer-readable program code adapted to be executed to implement one or more functions specified in the flow diagram block or blocks. The computer program instructions may also be loaded ono a computer or other programmable data processing apparatus to cause a series of operational elements or steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process such that the instructions that execute on the computer or other programmable apparatus provide elements or steps for implementing the functions specified in the flow diagram block or blocks. 
     Accordingly, blocks of the block diagrams and flow diagrams support combinations of means for performing the specified functions, combinations of elements or steps for performing the specified functions and program instruction means for performing the specified functions. It will also be understood that each block of the block diagrams and flow diagrams, and combinations of blocks in the block diagrams and flow diagrams, can be implemented by special-purpose, hardware-based computer systems that perform the specified functions, elements or steps, or combinations of special-purpose hardware and computer instructions.