Abstract:
The present invention provides a system and method for collection, storage and processing of tissues and cells. The system includes a collection container with chambers for storing and processing tissues, which are controllably separated and maintain a physiologic environment for the tissues. The system also includes a fluidic device for isolating target cells of interest. The method includes receiving the tissue into a collection chamber, transferring the tissue to a processing chamber, dissociating the tis sue into single cells, and passing the single cells to a device for isolating one or more target cells.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to PCT Application No. PCT/162014/064159, titled “MOLECULAR CELL IMAGING USING OPTICAL SPECTROSCOPY” and filed on Aug. 29, 2014, the entire contents of which are incorporated herein by reference. 
     
    
     FIELD 
       [0002]    The present disclosure relates to systems and methods for the storage and processing of surgical tissue samples into single cells and subsequent analysis using optical spectroscopy. 
       BACKGROUND 
       [0003]    Brain tumors are abnormal cell proliferations that occur in the central nervous system (CNS). It is estimated that there are over 23,000 new brain tumor cases in the United States resulting in over 14,000 deaths per year (Ostrom et al., 2013). Glioblastoma Multiforme (GBM), World Health Organization grade IV astrocytoma, is the most common and aggressive primary brain tumor in humans accounting for over 45% of all malignant brain tumors in the US (Ostrom et al., 2013). The current standard care for GBM involves a combination of chemotherapy with the oral methylating agent, temozolomide, radiation therapy, and/or maximal surgical resection. Although tumor shrinkage is observed following such treatments, brain tumor relapse is observed in around 90% of patients, resulting in a median survival of only 12 to 15 months (Stupp et al., 2009; Weller et al., 2013). 
         [0004]    There is evidence that cancer is maintained and driven by stem-like cells known as cancer stem cells (CSCs), similar to organs where maintenance and homeostasis are driven by adult stem cells (Beck and Blanpain, 2013; Zhou et al., 2009). CSCs were first isolated from leukemia, and have since been isolated from many solid tumors including breast, colon, pancreatic, prostate, skin, head and neck, ovarian, lung and liver tumors (Al-Hajj et al., 2003; Bapat et al., 2005; Bonnet and Dick, 1997; Collins et al., 2005; Curley et al., 2009; Dalerba et al., 2007; Eramo et al., 2008; Fang et al., 2005; Kim et al., 2005; Lapidot et al., 1994; Monzani et al., 2007; O&#39;Brien et al., 2007; Patrawala et al., 2006; Ponti et al., 2005; Quintana et al., 2008; Ricci-Vitiani et al., 2007; Schatton et al., 2008; Szotek et al., 2006; Vermeulen et al., 2008; Yang et al., 2008; Zhang et al., 2008). Brain tumor stem cells (BTSCs) were first isolated from post-operative brain tumor samples, including GBM, by sorting for surface markers that enriched for BTSCs in the CD133+ fraction (Singh et al., 2003; Singh et al., 2004). BTSCs exhibit properties of stem cells including their ability to self-renew in vitro as non-adherent neurospheres and multipotency in vitro, exhibited by the ability to differentiate into the three neural lineages including neurons, astrocytes, and oligodendrocytes. BTSCs also exhibit the same properties in vivo where the injection of as few as 100 CD133+ cells intracranially into immunodeficient xenograft models are able to reinitiate brain tumors that phenocopy the original patient, demonstrating multipotency of the BTSCs in vivo but more importantly, the ability of BTSCs to reinitiate the brain tumor. Finally, BTSCs exhibit self-renewal properties in vivo as CD133+ BTSCs can be isolated from the brain tumors of primary xenografts and serially transplanted into secondary xenografts and reinitiate brain tumor formation. These results demonstrate that a small population of cells within the brain tumor exhibit stem cell properties that allow these cells to initiate brain tumor formation. Note that the term CSCs may have different nomenclature in the field such as, but not limited to, tumor stem cells, tumor initiating cells, tumor progenitor cells, cancer initiating cells, or cancer progenitor cells. In this disclosure, the term CSCs encompasses all the cell types aforementioned. Similarly, this is also extended to BTSCs where the term brain tumor can be used as a prefix of the aforementioned terms to describe CSCs within brain tumors. 
         [0005]    The existence of BTSCs may explain the high recurrence and mortality rates seen in brain tumor patients who have undergone standard care (Stupp et al., 2009; Weller et al., 2013). One of the characteristics of CSCs is that they are able to evade many standard care treatments. For example, BTSCs have been shown to exhibit resistance to common antineoplastic chemotherapeutic drugs (Chen et al., 2012; Eramo et al., 2006) and to radiation therapy via preferential upregulation of the DNA damage checkpoint response and increase in DNA repair capacity (Bao et al., 2006). This preferential chemo- and radiation-therapy resistance is not unique to CSCs of the brain but has also been shown for CSCs of breast, colon, ovarian, pancreas, and leukemia (Adikrisna et al., 2012; Alvero et al., 2009; Diehn et al., 2009; Dylla et al., 2008; Kreso et al., 2013; Li et al., 2008; Oravecz-Wilson et al., 2009; Tehranchi et al., 2010; Todaro et al., 2007). 
         [0006]    Surgical procedures may not be able to target the removal of CSCs of the tumor apart from removing the bulk tumor itself. Therefore, the therapeutic resistance of CSCs is one mechanism by which tumor relapse may occur, as standard treatments are unable to target and remove CSCs, leaving them behind in patients. The residual CSCs are then able to reinitiate a tumor through their stem cell characteristics (self-renewal and multipotency) and lead to recurrence. Consequently, it would be beneficial to be able to distinguish CSCs from other tumor cells because eradication of the CSCs may be required to eliminate the cancer. In this context, the term distinguish refers to the ability to create contrast or identify one cell type, such as CSCs, from another, such as non-CSCs, bulk tumor cells, adult stem cells, or healthy tissue. 
         [0007]    Optical spectroscopy may be used to identify target cells such as CSCs. The optical absorption and scattering properties of biological tissue depend on both the chemical and structural properties of the tissue and the wavelength of the interacting light. How these absorption and scattering properties of tissue change as a function of light can be particularly useful, as it is often unique to chemicals or structures in the tissue (the spectrum of the tissue). For example the absorption features of oxy- and deoxy-hemoglobin can be used to measure the oxygenation of blood and tissue, and the scatter changes caused by different cellular sizes can be used to detect precancerous and cancerous tissue. The field of measuring these changes in optical properties as a function of light is known as spectroscopy and the device to measure the light at the various wavelengths is known as a spectrometer. Spectroscopy has found a wealth of current and potential applications in medicine. 
         [0008]    An example of optical spectroscopy is Raman spectroscopy, a rapid and nondestructive method to analyze the chemistry of a given material using light (Raman and Krishnan, 1928). Raman spectroscopy takes advantage of an optical property known as inelastic scattering that occurs when light interacts with matter. This inelastic scattering is unique to the molecular structures of the matter, thus providing a unique spectrum (or signature) of the matter that can be unambiguously distinguished and identified. Raman spectroscopy may provide neurosurgeons with an unambiguous and objective method to create contrast between tissues that are relevant to neurosurgery. For example, Raman spectroscopy may aid and assist neurosurgeons in distinguishing between healthy and tumor tissues, therefore minimizing the amount of tumour tissues left behind while preserving the critical healthy tissues, ultimately improving the surgical outcome of the patient. Studies using xenograft mouse models with transplanted brain tumor cells (Ji et al., 2013; Karabeber et al., 2014; Uckermann et al., 2014) and frozen human brain tumor sections (Kalkanis et al., 2014; Kast et al., 2014) have provided proof-of-principle of the potential of Raman spectroscopy in distinguishing healthy and tumor tissue. Raman measurements have been acquired from a number of different stem cell types ex vivo (Harkness et al., 2012; Hedegaard et al., 2010) but have not yet been acquired from BTSCs or from CSCs in vivo. 
         [0009]    Surgical removal of brain tumors may be done using port-based surgery. Port-based surgery is a minimally invasive surgical technique where a port is introduced to access the surgical region of interest using surgical tools. Unlike other minimally invasive techniques, such as laparoscopic techniques, the port diameter is larger than the tool diameter. Hence, the tissue region of interest is visible through the port. 
         [0010]    Tissue removal devices, such as the Myriad system (NICO Corp.), are commonly used to remove tissues from patients during port-based surgeries. Tissue removal devices typically store the removed tumor samples in a collection container connected to the tissue removal probe or surgical resector. In most cases, the stored tissue sample remains in the collection container until the end of the surgery before it gets processed at a remote laboratory. Therefore, there is a significant delay between the time the tissue sample is resected during surgery and when it gets processed. Furthermore, although the collection container is completely enclosed, the environment in the container does not mimic the in vivo environment. The delay in processing combined with the lack of proper in vivo storage can significantly impact the tissue sample&#39;s biology. 
         [0011]    What is lacking in the field is a way to visualize and remove target cells such as CSCs intraoperatively, and to store and isolate target cells in a way that maintains their in vivo characteristics. 
       SUMMARY 
       [0012]    In this disclosure, a method and system is described to provide intraoperative storage of resected tissue samples that mimics in vivo conditions, enable efficient processing of tissue samples into single cells and distinguish target cells from non-target cells. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  is a flow chart that depicts the steps involved in port-based neurosurgery and the harvesting, storage, processing, probing, and sorting of the tissue into single cells in an intraoperative manner. 
           [0014]      FIG. 2  is a schematic of a view down a port during neurosurgery. 
           [0015]      FIG. 3  is a schematic that illustrates the methods and barriers involved in isolating BTSCs from brain tumors. 
           [0016]      FIG. 4 a    is a schematic of the tissue container for the processing of tissue samples into single cells and the fluidic device for the probing and sorting of single cells. 
           [0017]      FIG. 4 b    is a schematic of the mixing channel section of the fluidic device with the purpose to move cells from digestive enzymes in one channel to cell culture media in another channel. 
           [0018]      FIG. 5  is a schematic illustrating the simultaneous use of a Raman microscope with a fluidic device. 
       
    
    
     DETAILED DESCRIPTION 
       [0019]    Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure. 
         [0020]    As used herein, the terms, “comprises” and “comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms, “comprises” and “comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. 
         [0021]    As used herein, the term “exemplary” means “serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein. 
         [0022]    As used herein, the terms “about” and “approximately” are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions. In one non-limiting example, the terms “about” and “approximately” mean plus or minus 10 percent or less. 
         [0023]    As used herein, the term in situ means in the tissue of origin; the term in vivo means within a living organism and refers to the location of tissues and/or cells in their native environment in the body. This in vivo location contains the environmental factors that are most ideal and/or suitable for the preservation of the tissue and/or cell biology; the term ex vivo means outside of a living organism; the term in vitro means within a culture dish, test tube or elsewhere outside a living organism; “target cells” means cells that are intended for identification or isolation, note that there could be multiple target cells simultaneously that are of interest for identification or isolation; “non-target cells” means cells that are not intended for identification or isolation; “surrounding tissue” means tissue outside of the tissue being measured; “adjacent cells” means cells within the same tissue as the cells being measured; “Raman Spectroscopy” includes fiber-based Raman systems incorporating transmissive grating or reflective grating, other variations of Raman spectroscopy including but not limited to Coherent anti-stokes Raman Spectroscopy (CARS), Shifted-excitation Raman difference spectroscopy (SERDS) and stimulated Raman Spectroscopy (SRS) and non-fiber based Raman systems; “Fluidic device” refers to any fluidic system, including a microfluidic system, that makes use of fabricated channels as a method to manipulate, control, transport fluid and/or cells through the use of passive capillary forces, or active forces, such as fluidic pumps, micropumps, or valves. 
         [0024]    In this patent, the term distinguish refers to the ability to create contrast or identify one cell type, such as CSCs, from another, such as non-CSCs, bulk tumor cells, adult stem cells, or healthy cells. 
         [0025]      FIG. 1  is a flow chart illustrating the processing steps involved in a port-based surgical procedure. The example here describes identification of BTSCs but those skilled in the art will recognize that the method can be applied to other target cells, such as, but not limited to, other CSCs. 
         [0026]    Surgical procedures are well known in the art. A first step involves importing a port-based surgical plan  101 . An exemplary plan may include preoperative 3D imaging data (i.e., MRI, ultrasound, etc.), overlaying received inputs (i.e., sulci entry points, target locations, surgical outcome criteria, additional 3D image data information) on the preoperative 3D imaging data and displaying one or more trajectory paths based on the calculated score for a projected surgical path. An example of a process to create and select a surgical plan is outlined in the disclosure “PLANNING, NAVIGATION AND SIMULATION SYSTEMS AND METHODS FOR MINIMALLY INVASIVE THERAPY”, International Patent Application CA2014050272 which claims priority to U.S. Provisional Patent Application Ser. Nos. 61/800,155 and 61/924,993, which are hereby incorporated by reference in their entirety. The aforementioned surgical plan may be one example; other surgical plans and/or methods may also be envisioned. 
         [0027]    Once the plan has been imported into the navigation system  101 , the subject is affixed into position using a head or body holding mechanism. The head position is also confirmed with the subject plan using the navigation software  102 . 
         [0028]    The next step is to initiate registration of the subject  103 . The phrase “registration” or “image registration” refers to the process of transforming different sets of data into one coordinate system. Data may be multiple photographs, data from different sensors, times, depths, or viewpoints. The process of “registration” is used in the present application for medical imaging in which images from different imaging modalities are co-registered. Registration is necessary in order to be able to compare or integrate the data obtained from these different modalities. 
         [0029]    Those skilled in the art will appreciate that there are numerous registration techniques available and one or more of them may be used in the present application. Non-limiting examples include intensity-based methods which compare intensity patterns in images via correlation metrics, while feature-based methods find correspondence between image features such as points, lines, and contours. Image registration algorithms may also be classified according to the transformation models they use to relate the target image space to the reference image space. Another classification can be made between single-modality and multi-modality methods. Single-modality methods typically register images in the same modality acquired by the same scanner/sensor type, for example, a series of MR images can be co-registered, while multi-modality registration methods are used to register images acquired by different scanner/sensor types, for example in MRI and PET. In the present disclosure multi-modality registration methods are used in medical imaging of the head/brain as images of a subject are frequently obtained from different scanners. Examples include registration of brain CT/MRI images or PET/CT images for tumor localization, registration of contrast-enhanced CT images against non-contrast-enhanced CT images, and registration of ultrasound and CT. 
         [0030]    Once registration is confirmed  104 , the subject is draped  205 . Typically draping involves covering the subject and surrounding areas with a sterile barrier to create and maintain a sterile field during the surgical procedure. The purpose of draping is to eliminate the passage of microorganisms (i.e., bacteria) between non-sterile and sterile areas. 
         [0031]    Upon completion of draping  105 , the next step is to confirm subject engagement points  106  and then prepare and plan craniotomy  107 . 
         [0032]    Upon completion of the preparation and planning of the craniotomy step  107 , the craniotomy is carried out  108  in which a bone flap is temporarily removed from the skull to access the brain. Registration data is updated with the navigation system at this point  109 . 
         [0033]    The next step is to confirm the engagement within the craniotomy and the motion range  110 . Once this data is confirmed, the procedure advances to the next step of cutting the dura at the engagement points and identifying the sulcus  111 . Registration data is also updated with the navigation system at this point  109 . 
         [0034]    In an embodiment, by focusing the camera&#39;s gaze on the surgical area of interest, this registration update can be manipulated to ensure the best match for that region, while ignoring any non-uniform tissue deformation affecting areas outside of the surgical field. Additionally, by matching overlay representations of tissue with an actual view of the tissue of interest, the particular tissue representation can be matched to the video image to ensure registration of the tissue of interest. For example, the embodiment can:
       Match video of post craniotomy brain (i.e. brain exposed) with imaged sulcal map;   Match video position of exposed vessels with image segmentation of vessels;   Match video position of lesion or tumor with image segmentation of tumor; and/or   Match video image from endoscopy up nasal cavity with bone rendering of bone surface on nasal cavity for endonasal alignment.       
 
         [0039]    In other embodiments, multiple cameras may be used and overlaid with tracked instrument(s) views, and thus allow multiple views of the data and overlays to be presented at the same time, which may provide even greater confidence in a registration, or correction in more dimensions/views than provided by a single camera. 
         [0040]    Thereafter, the cannulation process is initiated  112 . Cannulation involves inserting a port into the brain, typically along a sulci path as identified in step  111 , along a trajectory plan. Cannulation is an iterative process that involves repeating the steps of aligning the port on engagement and setting the planned trajectory  113  and then cannulating to the target depth  114  until the complete trajectory plan is executed  112 . 
         [0041]    The surgeon then performs resection  115  to remove part of the brain and/or tumor of interest. Resection  115  is a continual loop including both fine and gross resection  116 . During resection, the surgeon makes use of a resection tool within the port as described above and as further illustrated in  FIG. 2 . The port  201  is inserted during the cannulation process which provides the surgeon with a view of the tissue lying beneath. This tissue could represent both the tumor area  202  and/or the healthy area  203  separated by a tumor boundary  204 . A portion of the tissue beneath the port may also represent the location of the BTSCs  205  which the surgeon does not know a priori. During resection, a surgeon typically will use a resector tool  206  that has two functions, the first of which is to suction the tissue within the tool, and the second of which is to resect the tissue within the tool. As an addition, this is described in PCT Application No. PCT/162014/064159 “MOLECULAR CELL IMAGING USING OPTICAL SPECTROSCOPY”, optical spectroscopy can be used in conjunction  207  with the resector tool allowing targeted isolation of target tissue or cells. 
         [0042]    A problem that remains to be solved is a way to identify and isolate target cells, and in particular BTSCs intraoperatively. To solve this problem, four barriers to overcome are: i. identification of BTSCs, non-BTSC tumor cells, and healthy cells intraoperatively; ii. tissue resection in a minimally invasive manner in order to preserve the biology of the resected tissue and cells within; iii. storage of the tissues and cells within in a manner that mimics their in vivo environment to preserve their biology; and iv. isolation of target cells, such as BTSCs, from non-target cells, such as non-BTSC tumor cells, from the resected tissue. 
         [0043]    Regarding the first barrier,  FIG. 3  illustrates the current methodologies used to surgically remove brain tumor samples, which are a source of BTSCs. The workflow begins with a subject, such as a patient, exhibiting a brain tumor  301  and to be treated using surgical resection  302  to remove the tumor. Resection  302  of brain tumor is largely done in a non-targeted fashion. Available tools to neurosurgeons for removing brain tumors include using preoperative images, such as MRI, which become increasingly inaccurate intraoperatively as the brain shifts in position relative to the skull during surgery. Neurosurgeons also commonly use color contrast to distinguish between healthy and tumor tissue, which is highly subjective. Ultimately, the removal of brain tumor is largely performed using non-targeted and non-quantitative methods. For this reason, the extracted brain tumor  303  is largely a heterogeneous population of cells that consists of both tumor mass cells  304  that make up the majority of the brain tumor and BTSCs  305  propagating the brain tumor. Note that it is likely that residual BTSCs are also left behind during neurosurgical removal of the brain tumor. Thus, the first barrier  306  relates to the limitations of current methods to visualize and distinguish target tissues or cells, such as BTSCs, intraoperatively. The first barrier of BTSC identification in situ is described in patent application PCT Application No. PCT/162014/064159 “MOLECULAR CELL IMAGING USING OPTICAL SPECTROSCOPY”. 
         [0044]    Regarding the second barrier, once the target tissue or cell has been defined via imaging  306 , the target tissue or cell is resected  302 . The BTSC biology can be altered during procedures of brain tumor resection such as intraoperative manipulation, extraction technique and handling. Therefore, traditional devices such as ultrasonic aspirators and coagulation instruments that cause dissipation of thermal energy not only damage surrounding healthy brain tissue but may also compromise BTSCs&#39; biology (McLaughlin et al., 2012). Hence, minimal manipulation of BTSCs intraoperatively and the use of nonablative instrumentation is preferred to preserve BTSC physiology. An example of a non-ablative instrument for tissue resection is the Myriad System (NICO Corp.). The Myriad system includes a resector tool which allows the isolation of tissue without crushing, or thermal and ablative damage on the sample, thereby preserving the tissue&#39;s biology (McLaughlin et al., 2012). 
         [0045]    Regarding the third barrier, returning to  FIG. 3 , once a brain tumor sample  303  has been identified  306  in situ and resected  302 , the tumor sample  303  is usually transported to a collection container connected to the tissue removal probe or surgical resector. In most cases, the stored tissue remains in the collection container until the end of the surgery before it goes through procedures for tissue storage and/or tissue processing  307 . In the case of neurosurgery, the time between the start of tissue removal and the end of surgery may be in the order of hours, which is detrimental to the tissue&#39;s biology. Furthermore, although the collection container is completely enclosed, the environment in the container does not mimic the in vivo environment. For example, exposure to an atmosphere where the temperature, pH, oxygen conditions, and/or growth factors can vary from the tissue&#39;s native niche can alter the tissue&#39;s biology, genetics, epigenetics, chemistry, and metabolism instantaneously inducing BTSC death and differentiation (Bar et al., 2010; Soeda et al., 2009; Zhou et al., 2011). For these reasons, systems and methods that can store  307  tissues in a manner that preserves the tissue biology before the tissue is processed, followed by rapid processing  307  intraoperatively is a current unmet market need. In the case of brain tumors, the rapid processing and interrogation of brain tumor samples is important as the median survival for GBM patients is in the order of months. Rapid processing of a patient&#39;s brain tumor sample can provide important insights into, for example, their treatment regimen in a timely manner. 
         [0046]    Returning to  FIG. 1 , a method for processing resected tissue is provided which overcomes the limitations of the current art. The resected tissue is collected  117  into a tissue container and stored under physiological conditions, as described in detail below. The tissue remains in the tissue container until the process chamber is ready  118 . When the process chamber is ready  118 , the tissue is moved to the process chamber, where the tissue is dissociated into single cells  119  using enzymatic and physical manipulation. Dissociated cells are then separated into cell clumps which are passed into a storage container and single cells which are passed into a fluidic system (described in detail below). Within the fluidic system, the cells are probed by optical spectroscopy  120 . Based on spectral measurements, the cells are sorted  121  and stored  122 . 
         [0047]    Once resection is complete  115 , the tissue is decannulated  123  by removing the port and any tracking instruments from the brain. Finally, the surgeon closes the dura and completes the craniotomy  124 . 
       DETAILED DESCRIPTION OF FIG.  4   
       [0048]    As seen in  FIG. 4 a   , the system and method for storing, processing and separating tissue includes a tissue processing container  401  which is provided with a temperature and humidity controller  402 . The tissue processing container  401  includes a collection chamber  403 , a process chamber  404  and a waste chamber  405 . The collection chamber  403  is separated from the process chamber  404  by a controllable separator  406 . The process chamber  404  is separated from the waste chamber  405  by a controllable solid filter  407 . 
         [0049]    The collection chamber  403  is connected to a resector tool  408  through a collection tube  409 . The collection chamber  403  is also connected to a gas controller  410  through a gas inlet  411 , and a media dispenser  412  through a media inlet  413 . 
         [0050]    The process chamber  404  is provided with rotatable blades  414  which are connected through a shaft  415  to a blade motor  416 . The process chamber  404  is connected to a saline dispenser  417  through a saline inlet  418  and a digestive enzymes dispenser  419  through a digestive enzymes inlet  420 . A cell outlet  421  leads from the process chamber  404  to a fluidic device  422  and a cell storage outlet  423  leads from the process chamber  404  to a first cell storage container  424 . 
         [0051]    The waste chamber  405  is connected to an excess fluid container  425  through an excess fluid outlet  426 . 
         [0052]    The fluidic device  422  includes a fluidic buffer  427  that converts a large fluidic channel to a small fluidic channel, a single cell filter  428  and a temperature control plate  429 . The fluidic device  422  is connected to a first fluidic pump  430  and a media exchange reservoir  431  through a media inlet  432 . Multiple channels  433  connect the fluidic device  422  to multiple cell storage containers  434 , which are connected to a second fluidic pump  435 . 
         [0053]    The fluidic device is also connected to a laser  436  through a fiber bundle including excitation fibers and detection fibers. 
         [0054]    The laser  436 , gas controller  410 , first fluidic pump  430 , media dispenser  412 , PBS dispenser  417 , digestive enzyme dispenser  419 , cell outlet  421 , first cell storage outlet  423 , temperature and humidity controller  402 , and blade motor  416  are electronically connected to a control box  437 . 
         [0055]    During port-based surgery, the resector tool  408  is used to perform resection as described in  FIG. 1  above. The resected tissue sample  438  is collected into the tissue processing container  401  via the collection tube  409 . The tissue processing container  401  is an enclosed and sterile system. The environment of the tissue processing container  401  is customized by the temperature and humidity controller  402 , and the gas controller  410  for nitrogen, oxygen, and carbon dioxide to control oxygen tension. The internal surface of the tissue processing container  401  may also be coated with (ECM), such as collagen, laminin, fibronectin or poly-L-ornithine, and have a 3D culture surface to further simulate in vivo conditions. 
         [0056]    When the resected tissue sample  438  arrives in the tissue processing container  401 , it is first collected in the collection chamber  403 . The collection chamber  403  serves as an area where the resected tissue sample  438  is collected intraoperatively as surgery proceeds and stored before being processed. During this time, the biology of the tissue sample  438  can be preserved by modulating the variables (temperature, humidity, oxygen tension, ECM) mentioned previously to mimic the in vivo environment., For example, for BTSC, the ideal physiological temperature and humidity may be 37° C. and 95%, respectively, along with 5% CO 2  and 5%˜21% O 2 . In addition, specific cell culture media  412  may be added into the collection chamber  403  which can further provide the tissue with favorable conditions to preserve its biology. For example, the use of favorable conditions that promote self-renewal of normal neural stem cells (NSCs) such as the use of growth factors including Fibroblast Growth Factor 2 (FGF2) and Epidermal Growth Factor (EGF), and ECM including laminin and Poly-L Ornithine, may help preserve BTSC biology. 
         [0057]    In the collection chamber  403 , the tissue samples are continually being collected and stored before processing. If the process chamber  404  is not ready for receiving the tissue sample  438 , the controllable separator  406  remains in the closed position, preventing the tissue sample  438  from proceeding to the next stage. For example, the processing chamber  404  could be not ready because it is currently processing other tissue samples. This dual collection  403  and processing  404  chamber allows tissue samples to be collected and processed simultaneously. If there are no tissue samples being processed in the processing chamber  404 , the process chamber  404  is ready to receive the tissue sample  438  from the collection chamber  403 , and the controllable separator  406  opens, allowing the tissue sample  438  to drop to the processing chamber  404 . 
         [0058]    The role of the processing chamber  404  is to dissociate the tissue sample  439  into single cells. To achieve this, the controllable solid filter  407  opens to allow excess fluid  440  including, but not limited to, cell culture media, blood, and cerebral spinal fluid, to the waste chamber  405 , while preventing solids, such as the tissue sample  439 , from passing through. Once the processing chamber  404  is, devoid of liquids, the controllable solid filter  407  closes to prevent any more liquid from passing through. To prepare the tissue sample  439  for dissociation, a saline solution, such as Phosphate Buffer Saline (PBS)  417 , is added to the processing chamber  404  to submerge the tissue sample  439 . The blade  414  rotates to aid in the mixing and washing of the tissue sample  439  with PBS. After washing for 5 to 15 minutes, the blade  414  stops rotating, the controllable solid filter  407  opens to allow the used PBS to flow through, and then the controllable solid filter  407  closes again. This washing process can be repeated multiple times, such as up to three times, to ensure thorough washing of the tissue. Once the washing step is complete, the processing chamber  404  is emptied of excess fluids, and the controllable solid filter  407  is closed, then the tissue sample  439  is ready for dissociation. The tissue sample  439  is dissociated by the addition of digestive enzymes  419  such as, but not limited to, trypsin, collagenase, or accutase, into the processing chamber  404  sufficiently to submerge the tissue sample  439 . Once the digestive enzyme is added, the blade  414  is turned on to aid in the mixing and dissociation of the tissue sample  439 . The time required for digestive enzymes to dissociate tissue samples  439  into single cells varies with the digestive enzyme agent used and the size of the tumor sample. Generally, the process takes from anywhere between  15  minutes to an hour, but could also be beyond these time ranges. Note that the processing chamber  404  is also subjected to the same environmental controllable variables described for the collection chamber  403  described above (temperature, humidity, oxygen tension, ECM) to mimic the in vivo environment. 
         [0059]    Once the dissociation step is complete, the single cells can be sent for further processing (described below). While the tissue sample  439  is being processed in the processing chamber  404 , resected tissue samples  438  continue to be collected in the collection chamber  403 . After the dissociation step is complete and the processing chamber  404  is devoid of tissues  439  or single cells, the next round of tissue samples  438  is deposited into the processing chamber  404  by opening the controllable separator  406  and the dissociation step is repeated. For these reasons, the tissue collection step and the tissue dissociation step can occur continuously and simultaneously throughout the surgical procedure without interruption. 
         [0060]    The excess fluids  440  which include, but are not limited to, cell culture media, blood and cerebral spinal fluid, may be of significance for research purposes. For example, exosomes found in the serum of blood have significant roles in tumor pathogenesis (Abd Elmageed et al., 2014) and may serve as important diagnostic and prognostic factors. Therefore, it is advantageous to collect the excess fluid  440  into a container  425 , which can then be used for downstream analysis (described below). 
         [0061]    Returning to  FIG. 3 , once the brain tumor  303  has been stored and processed  307  into single cells  308 , the fourth barrier relates to the ability to isolate  309  the BTSCs  310  from the non-BTSCs. In this context, non-BTSCs can include, healthy cells, non-BTSC tumor cells, and/or normal NSCs. Current state of the art to establish CSC lines  310  from tumor samples  303  include multiple methods of isolation  309  techniques. As an example, current methods for the isolation of BTSCs from brain tumors are provided here. 
         [0062]    Isolation  309  of BTSCs  305  from a heterogeneous population of brain tumor  303  includes the use of cell surface markers such as CD 133  for sorting through flow cytometry (Singh et al., 2004) and the use of favorable conditions that promote self-renewal of normal NSCs such as providing growth factors including FGF2 and EGF, ECM including laminin and Poly-L Ornithine (Pollard et al., 2009), and hypoxic oxygen concentrations such as 5% oxygen. The use of these techniques allows the isolation of BTSCs  305  from non-BTSCs  304  in the brain tumor sample  303 . 
         [0063]    Once BTSCs are isolated from the tumor cells, they are expanded. To expand newly isolated BTSCs  310 , BTSCs can be propagated in vitro by several methods including 1) non-adherent or 2) adherent methods. In the non-adherent method, BTSCs are typically propagated in a low attachment container in the favorable conditions described above (growth factors and oxygen concentrations) promoting BTSCs to adhere to each other rather than the container and form spheres of cells known as neurospheres. These neurospheres can then propagate and expand in this configuration. In the adherent method, BTSCs are typically propagated in a container coated with a favorable ECM, promoting their attachment to the container. BTSCs will then be propagated in favorable conditions described above (growth factors and oxygen tension). These BTSCs, described as monolayers, can then propagate and expand in this configuration. Once BTSCs are stably propagating in vitro, they are considered a BTSC line  310 . 
         [0064]    The current need to culture BTSCs in vitro during isolation  309  is problematic as the culturing of BTSCs  310  in vitro can impose artifacts, such as, but not limited to, genetic and epigenetic changes, such that the BTSCs  310  do not resemble when they were in their in vivo state. The current norm of studying BTSCs that have been cultured in vitro may impact and confound any opportunities  311  such as research  312  to be performed on such BTSCs, yielding data that may not be relevant to their in vivo counterparts. There is currently a need to culture BTSCs in vitro because there are no methods to directly isolate the BTSCs in situ during surgery  302 , the first barrier described above. This is described in PCT Application No. PCT/162014/064159 “MOLECULAR CELL IMAGING USING OPTICAL SPECTROSCOPY” the use of optical spectroscopy, such as Raman spectroscopy, to identify BTSCs in situ intraoperatively  306  during resection by comparing acquired spectra to a database of spectral signatures of known cell types. The use of optical spectroscopy to distinguish and isolate target cells, such as BTSCs, in situ can also be done after resection as a method to isolate  309  BTSCs  310  from a heterogeneous population of cells  308  intraoperatively. Note that it is also possible to utilize optical spectroscopy both during resection  306  and after processing  307  for the isolation  309  and confirmation of target cells  310 . 
         [0065]    Returning to  FIG. 4 a   , after the tumor sample has been dissociated into single cells, the single cells flow from the cell outlet  421  into the fluidic device  422 . Fluidic devices, such as continuous-flow fluidics, take advantage of a continuous liquid flow through fabricated channels. The liquid flow-through is driven by external pressure sources such as mechanical pumps, integrated mechanical micropumps, or a combination of capillary forces and electrokinetic mechanisms. 
         [0066]    The dissociated single cells in the processing chamber  404  flow  441  into the fluidic device  422  along with the fluid from the processing chamber. The fluidic device may also include multiple channels for the cells to flow into and within the fluidic device to allow more efficient processing of the cells. The fluidic device is attached to a temperature control plate  429  to maintain the ideal in vivo temperature, such as 37° C., for the single cells while the cells are in the fluidic device  422 . As the single cells flow into the fluidic device  422 , the single cells will flow into a fluidic buffer  427  that converts a large fluidic channel to a small fluidic channel in which the single cells flow in a file of single cells. The cells then flow through a single cell filter  428  to ensure any residual cells that are clumped together are redirected through a cell outlet  442  and to a cell storage container  443  for later use and do not hinder the rest of the fluidic device  422 . 
         [0067]    When the single cells have passed through the single cell filter  428 , they are still in digestive enzyme. Therefore, the cells will move into the mixing channel section  444  where the digestive enzyme will be removed and cell culture media will be added to preserve cell biology.  FIG. 4 b    illustrates the mixing channel section  445  in detail within the fluidic device  422  where single cells in digestive enzyme  446  enter through a fluidic channel. In an adjacent fluidic channel, cell culture media  447 , is input from the media exchange reservoir  431 ,  448 . The goal of the mixing channel section  445  is to move  449  the single cells flowing through the fluidic device  422  from flowing in the channel with digestive enzyme  446  to the channel with cell culture media  450 . The channel of digestive enzyme devoid of single cells  451  is then discarded. The movement of the single cells  449  may be performed using lasers from an external source  452  directed at a single cell to generate optical forces to push the single cell from the fluidic channel with digestive enzyme  446 ,  451  to the fluidic channel with cell culture media  447 ,  450 . 
         [0068]    The laser  452  generating optical forces to move  449  the single cells may also serve a dual purpose of interrogating the single cell. A preferred example where a laser can be used as both a cell sorter and identifier is Laser Tweezer Raman Spectroscopy (LTRS) (Chan et al., 2009; Chan et al., 2008). This technique combines the functionality of optical tweezers with that of confocal Raman spectroscopy into a single module, allowing the capture, identification, and sorting of cells to be done simultaneously with lasers. Optical tweezers enable a single cell to be captured in the focus area of the confocal Raman microscope which enables Raman acquisition on a single cell. After the Raman acquisition, the laser is directed on to the single cell along the plane of the fluidic device and optical forces from the laser move the single cell into a different channel. The laser may also be integrated into the fluidic device. 
         [0069]      FIG. 5  illustrates a Raman microscope  501  integrated with the fluidic device  502  allowing interrogation of cells intraoperatively. Fluidic devices are on the orders of centimeters in length, which may be placed on a confocal microscope in the operation room away from the patient. Therefore, this enables studies and interrogation of cells intraoperatively with both optical and non-optical methods. It is important to note that the probing  120  of the cells and the sorting  121  need not occur simultaneously, as in LTRS, but may occur sequentially with multiple laser sources  436 ,  452 . In this disclosure, probing  120  refers to the process of interrogating a cell&#39;s identity, such as, but not limited to, via optical spectroscopy, to determine whether it is a target cell of interest, for example, a BTSC, a non-BTSC tumor cell, healthy cell, or normal NSC. Sorting  121  refers to the process of separating the different target cell types after probing  120 , for example, in to multiple fluidic channels and/or to discard non-target cells. 
         [0070]    Returning to  FIG. 4 a   , once the single cells in the cell culture media have been probed, for example, by Raman spectroscopy, the Raman spectra generated can be compared to a database of Raman spectra of known cell types. If a cell&#39;s spectrum is the same as that of a target cell spectrum within the database or is within a pre-determined range, then the cell is identified as a target cell. Based on the spectra comparison, the target cells of interest, such as BTSCs and non-BTSCs, are sorted into multiple fluidic channels  433  which flow into separate storage containers  434 . Stored cells may be used for downstream applications, including, but not limited to, 1) direct implantation into immunodeficient mice; 2) long term storage in liquid nitrogen; 3) storage in containers  434  subject to the same environmental controllable variables described for the collection  403  and processing chamber  404  (temperature, humidity, oxygen tension, ECM); or 4) long term culture in the storage containers  434  similar to bioreactors by employing similar mechanics as the tissue processing container  401  described above. Finally, it is possible that not all the single cells from the processed tissue  439  will go through the fluidic device  422 . Therefore it is possible to store any excess tissue  439  or cells in a storage container  424  connected to the process chamber  404  for future use. 
         [0071]    In one example, fluids are moved through the fluidic device  422  by passive forces such as capillary forces. In another example the fluid may be moved through external forces such as active fluidics, for example fluidic pumps or micropumps  430 ,  435 . The entire system as illustrated in  FIG. 4 a    also involves multiple mechanics including the control of cell culture media  412 , saline  417 , digestive enzymes  419 , cell, tissue, and fluid storages  424 ,  425 ,  434 ,  443 , fluidic pumps  430 ,  435 , lasers  436 , gas  410 , and temperature and humidity  402 . A control box  437  may electrically control any or all of the multiple mechanics. 
         [0072]    As mentioned previously, the excess fluids  440  may be of significance for study. Therefore, the fluidic device  422  may also be used to interrogate the excess fluids  440  by optical spectroscopy techniques, such as Raman Spectroscopy for important factors, such as exosomes and other extracellular vesicles within the blood or cerebral spinal fluid of patients. 
       Verification of BTSCs 
       [0073]    Returning to  FIG. 3 , prior to the use of BTSCs for research purposes, it is important to verify  313  their stem cell properties and confirm their identity as BTSCs. Similar to other stem cells, BTSCs should possess the two properties of stem cells, self-renewal and multipotency. Both these properties can be demonstrated in vitro where self-renewal is demonstrated via the routine propagation of the BTSCs as neurospheres or as a monolayer described above. Multipotency, or the ability to differentiate, can be demonstrated in vitro by placing BTSCs in differentiation inducing conditions by removing them from the self-renewal conditions described above (growth factors, oxygen tension, and substrate). For example, BTSCs can be placed in media lacking self-renewing factors FGF2 and EGF with the presence of serum to promote their differentiation into the three neural lineages, neurons, astrocytes, and oligodendrocytes, which can then be confirmed by molecular techniques including, but not limited to, immunocytochemistry and quantitative polymerase chain reaction. It is also vital to verify the stem cell properties of BTSCs in an animal or in vivo, where in vivo means within a living organism. Typically, to perform in vivo characterization, a xenograft assay is done, that is, BTSCs are injected intracranially into another species  314 . The xenograft host is usually an immunocompromised rodent. Multipotency is demonstrated by the development of a brain tumor in the xenograft host by the injected BTSCs  310 . The brain tumor in the xenograft should reflect pathologically the original brain tumor  301  in the subject, demonstrating the BTSCs&#39; ability to differentiate into the different cell types comprising the original brain tumor. To demonstrate self-renewal in vivo, serial transplantation can be performed. This is demonstrated by isolating BTSCs from the brain tumor formed in the xenograft  310 , re-transplanting into a secondary xenograft recipient, and showing that a brain tumor can form again in the secondary xenograft. In theory, this can be performed over multiple serial transplantations demonstrating the self-renewal of BTSCs in vivo. Upon verifying that BTSC lines have these stem cell properties, they are suitable for future use, including research  312 , experimentation, and other opportunities  311 . 
       EXAMPLE 1 
       [0074]    An example is provided here of the collection, storage and processing of BTSC, although it is equally applicable to the collection, storage and processing of other cells, such as other cancer stem cells or other cell types. 
         [0075]    The collection, storage and processing is preferably carried out in the collection container described in  FIGS. 4 a  and 4 b   . In operation, the collection container is controlled to be approximately 37° C. and the humidity is maintained at approximately 95% by the humidity controller. 
         [0076]    Brain tumor tissue is resected using a resection tool, preferably as described in  FIG. 2 . The resected brain tumor tissue is collected through the collection tube to the upper collection chamber of the collection container and is stored there until the process chamber is empty and ready to receive tissue. Cell culture media that promote self-renewal of normal NSCs such as the use of growth factors including FGF2 and EGF, ECM including laminin and Poly-L Ornithine is added to the collection chamber from the cell culture media dispenser to cover the brain tumor tissue and a 5% CO 2  and 5% O 2  (hypoxic conditions) atmosphere is maintained by the gas controller. 
         [0077]    When the process chamber is empty and ready to receive a sample, the separator partitioning the collection chamber from the process chamber opens to allow the tumor sample to pass to the process chamber. The solid filter partitioning the process chamber from the waste chamber opens to allow the cell culture media and other tissue-associated fluids and small solids to pass through to the waste chamber beneath the process chamber. The solid filter then closes and PBS is dispensed from the PBS dispenser into the process chamber and the tumor sample is washed in the PBS by mixing with the rotatable blade. The wash step with PBS is continued for 15 minutes, then the controllable filter is opened to allow the PBS to move to the waste chamber and the controllable filter is closed again. The PBS wash step is repeated two more times. After the tumor sample is washed, collagenase enzyme is dispensed from the digestive enzymes container. The rotatable blade then slowly rotates for 1 hour to dissociate the brain tumor tissue into single cells. The opening and closing of the separator and solid filter, the dispensing of solutions and the rotation of the blades is controlled by a control box. 
         [0078]    After incubation in the enzyme solution and mixing with the rotatable blades to dissociate the tumor tissue into single cells, the cell outlet opens to allow passage of the cell suspension to the fluidic device. Cell movement to the fluidic device is maintained by a fluidic pump to bring the brain tumor cells into the fluidic device. The brain tumor cell suspension then continues to flow through the single cell filter, which partitions single cells from cell clumps. Cell clumps are diverted to a storage container. Single cells continue to move through the fluidic channel in the collagenase solution, until the channel parallels the cell culture containing channel. 
         [0079]    A laser connected to the trypsin-containing channel through an excitation fiber transmits optical waves at 785 nm, which generates a physical force that moves the cell to the adjacent cell culture channel. At the same time, a detection fiber receives the transmitted optical spectrum from the probed cell and carries it to the control box, where it is compared with a consensus BTSC spectrum. If the spectra of the interrogated cell and a consensus BTSC match within 10% of values, a subsequent laser connected by an excitation fibre emits optical waves that elicit a force to direct the cell to a channel leading to a storage container for targeted cells. Other cells are identified by the optical spectroscopy as tumor cells or non-tumor cells and are directed to separate channels leading to storage containers, allowing sorting of multiple categories of cells. The BTSC are stored in the storage container under ideal physiological conditions, namely 5% CO 2 , 5% O 2 , 95% humidity, in NSC promoting cell culture media until the surgery is complete. Cells are then divided into an aliquot for storage in liquid nitrogen, aliquot for in vitro culture, and an aliquot for xenograft assay into a mouse xenograft model. The mouse xenograft model is used to test drug therapies for effectiveness at BTSC death. 
         [0080]    The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.