WO2022173345A1 - An amniotic cell separating apparatus - Google Patents

An amniotic cell separating apparatus Download PDF

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Publication number
WO2022173345A1
WO2022173345A1 PCT/SE2022/050111 SE2022050111W WO2022173345A1 WO 2022173345 A1 WO2022173345 A1 WO 2022173345A1 SE 2022050111 W SE2022050111 W SE 2022050111W WO 2022173345 A1 WO2022173345 A1 WO 2022173345A1
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Prior art keywords
amniotic
cells
morphology
separation
ultrasound transducer
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PCT/SE2022/050111
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French (fr)
Inventor
Jan TALTS
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Amniotics Ab
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Publication of WO2022173345A1 publication Critical patent/WO2022173345A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
    • B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements

Definitions

  • the present invention pertains to an apparatus for separating amniotic cells in an amniotic fluid and a related method.
  • the amniotic fluid is the liquid surrounding and protecting the fetus during pregnancy. This fluid serves as a cushion for the growing fetus, but also serves to facilitate the exchange of nutrients, water, and biochemical products between mother and fetus. It is generated from maternal plasma, and passes through the fetal membranes by osmotic and hydrostatic forces. When fetal kidneys begin to function in about gestational week 16, fetal urine also contributes to the fluid. Amniotic fluid consists of water with electrolytes, but also contains proteins, carbohydrates, lipids, phospholipids, and urea. In addition to metabolic wastes, amniotic fluid also contains fetal cells and other materials chafed off the skin such as hair and vernix.
  • the amniotic fluid contains a number of valuable cells, including Mesenchymal stem cells (MSC).
  • MSC Mesenchymal stem cells
  • the MSC possess the characteristics of self-renewing and differentiating into multiple cell types, including osteoblast, adipocyte, chondrocyte, myocyte, and fibroblast.
  • the MSC:s can have a multilineage differentiation potential into bone, fat, and cartilage cells, and may thus be utilized for a variety of therapeutic applications, including personalized and regenerative medicine.
  • the amniotic fluid also contains terminally differentiated cells with limited proliferation capacity.
  • the MSC:s may be distinguished from other cells based on their morphology, or based on other characteristic such as genetic markers. It is thus possible to isolate stem cells with mesenchymal characteristics from the amniotic fluid.
  • the morphology of a cell refers to parameters such as the shape, structure, or size of the cell. Morphologically, MSC:s are fibroblast like and spindle-shaped cells. Sorting based on morphology is traditionally accomplished by standard isolation techniques involving e.g. repeated centrifugation and washing of the cell sample.
  • the available approaches and apparatuses for collecting, extracting, and isolating the stem cells of interest are not entirely satisfactory, for example, in their safety, avoidance of contamination (e.g., air contamination) of collected material, cell yield, efficiency, and/or ability to avoid destruction of components.
  • contamination e.g., air contamination
  • the quality of the stem cell samples may thus be negatively impacted, in particular in applications involving a large number of processing steps.
  • An objective is to at least partly overcome one or more of the above identified limitations of the prior art.
  • One objective is to provide an apparatus and method for high-yield, and high- precision, separation and isolation of desired amniotic cells such as stem cells with the potential for reprogramming, with minimal alteration or destruction of the cellular properties.
  • an amniotic cell separation apparatus comprising a fluidic cavity comprising an amniotic fluid sample inlet configured to connect to a sample source of amniotic fluid, the amniotic fluid containing amniotic cells and particulate matter, an ultrasound transducer arranged along the amniotic fluid sample inlet and being configured to generate an acoustic wave in the fluidic cavity, a controller connected to the ultrasound transducer, wherein the controller is configured to drive the ultrasound transducer with a frequency (f) that generates said acoustic wave, whereby when an amniotic fluid flows in the sample inlet, the acoustic wave forces amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel of the fluidic cavity, wherein the separation channel is connected to a
  • a method for separating amniotic cells in an amniotic fluid containing amniotic cells and particulate matter comprising passing the amniotic fluid through a sample inlet of a fluid cavity, and generating an acoustic wave in the fluidic cavity to force amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel of the fluidic cavity.
  • Some examples of the disclosure provide for an effective filtering of amniotic fluid.
  • Some examples of the disclosure provide for improved filtering of amniotic fluid containing a greater range in the size of particulate matter.
  • Some examples of the disclosure provide for obtaining stem cell samples of high quality.
  • Some examples of the disclosure provide for an efficient separation and isolation of stem cells of a desired morphology.
  • Some examples of the disclosure provide for a high-throughput isolation of stem cells from an amniotic fluid.
  • Some examples of the disclosure provide for a sterile isolation of stem cells from an amniotic fluid.
  • Some examples of the disclosure provide for a less complex apparatus for isolation of desired stem cells from of an amniotic fluid.
  • Some examples of the disclosure provide for a more compact apparatus for isolation of desired stem cells from of an amniotic fluid.
  • Some examples of the disclosure provide for obtaining an higher fraction of desired stem cells from a sample of amniotic fluid.
  • Fig. 1 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 2 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 3 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 4 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 5 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 6 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 7 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example
  • Fig. 8 is a flow chart of a method for separating amniotic cells in an amniotic fluid according to one example.
  • Fig. 1 is a schematic illustration of an amniotic cell separation apparatus 100 according to one example.
  • the amniotic cell separation apparatus 100 comprises a fluidic cavity 101.
  • the fluidic cavity 101 comprises an amniotic fluid sample inlet 102 configured to connect to a sample source 201 of amniotic fluid.
  • the apparatus 100 may comprise an inlet connector 112 arranged to sealingly connect the sample inlet 102 to the amniotic fluid sample source 201 .
  • the amniotic fluid contains amniotic cells, such as stem cells, e.g. Mesenchymal stem cells (MSC).
  • the amniotic fluid also contains terminally differentiated cells, e.g. keratinocytes, and other materials chafed off the skin such as hair and vernix.
  • the amniotic fluid thus generally contains a mixture of amniotic cells and particulate matter.
  • the amniotic fluid may thus flow along the sample inlet 102 being a fluidic channel which extends along a longitudinal direction (L) as indicated in e.g. Fig. 1.
  • the amniotic cell separation apparatus 100 comprises an ultrasound transducer 103 arranged along the amniotic fluid sample inlet 102.
  • the ultrasound transducer 103 is configured to generate an acoustic wave in the fluidic cavity 101.
  • the amniotic cell separation apparatus 100 may comprise ultrasound transducer’s 103 arranged at opposite sides of the amniotic fluid sample inlet 102, along a transverse direction (r) perpendicular to the longitudinal direction (L) of the sample inlet 102, as schematically illustrated in the example of Fig. 1.
  • amniotic cell separation apparatus 100 may comprise any number of ultrasound transducers 103, and that the position of the ultrasound transducer(s) 103 relative to the sample inlet 102 can be varied to optimize the characteristics of the acoustic wave in the fluidic cavity 101 and in the sample inlet 102 thereof.
  • Figs. 3 - 5 are schematic illustrations of further examples of the amniotic cell separation apparatus 100 which will be described further below.
  • the amniotic cell separation apparatus 100 comprises a controller 104 connected to the ultrasound transducer 103.
  • the controller 104 is configured to drive the ultrasound transducer 103 with a frequency (f) that generates the aforementioned acoustic wave.
  • a frequency (f) that generates the aforementioned acoustic wave.
  • the acoustic wave, or acoustic field, generated by the ultrasound transducer 103 creates acoustic pressure nodes across the width of the sample inlet 102, in the transverse direction (r), because of the acoustic vibrations.
  • the amniotic cells and the particulate matter will be exposed to a net force due to the pressure nodes which is dependent on the physical properties of the amniotic cells and particulate matter relative to the surrounding medium in which the amniotic cells and particulate matter are suspended.
  • the force exerted onto the amniotic cells or particles is also referred to as the acoustic radiation force.
  • the direction of the force is dependent on the relationship between the physical properties of the amniotic cells or particles and the surrounding medium.
  • the relationship can be expressed in the form of an acoustophoretic contrast factor (f).
  • the factor f is determined by the relative compressibilities and the densities of the medium and the cell/particle.
  • Amniotic cells having different morphologies, i.e. different physical properties, can thus have different associated acoustophoretic contrast factors (f).
  • the direction of movement of the amniotic cells relative to the pressure nodes which extend in the transverse direction (r) thereby depends on the acoustophoretic contrast factor (f) of the particular amniotic cell.
  • the different amniotic cells may thus be displaced in different directions along the transverse direction (r).
  • amniotic cells having a positive acoustophoretic contrast factor (f > 0) may be displaced in a first direction such as towards the transverse direction (r) illustrated in Fig. 2 (vertically upwards in Fig. 2), while amniotic cells having a negative acoustophoretic contrast factor (f ⁇ 0) may be displaced in a second direction, such as opposite the first direction (vertically downwards in Fig. 2).
  • the schematic illustration of Fig. 2 thus shows an example where cells of different morphologies are separated along the transverse direction (r) as the amniotic fluid flows along the longitudinal direction (L).
  • Amniotic cells of a first morphology such as MSC:s, are separated and collected in a separation channel 105 of the fluidic cavity 101 .
  • the separation channel 105 is connected to a first outlet connector 106 to sealingly connect the separation channel 105 to an amniotic cell-receiving device 202, where the amniotic cells of the first morphology may be collected.
  • the amniotic cells of the first morphology may in one example comprise extracellular vesicles (EV’s). Extracellular vesicles are present in the amniotic fluid as lipid bilayer-delimited particles that are naturally released from a cell, and may have desirable properties for therapeutic purposes, such as delivering nucleic acids or other cargo to diseased tissue.
  • amniotic cells and particles having different morphologies and characteristics are displaced different amounts in the transverse direction (r) to control the respective flow trajectories in the fluidic cavity 101 , for separation and isolation of the desired amniotic cells.
  • This provides for an effective separation of stem cells of interest, such as MSC:s, from an amniotic fluid sample source.
  • the amniotic fluid may contain a relatively low concentration of stem cells, such as MSC:s.
  • the concentration may be 1 cell/mL amniotic fluid for the MSC:s.
  • the separation allows for increasing the aforementioned concentration in the cell sample obtained by the amniotic cell separation apparatus 100.
  • Stem cell samples of a desired concentration of stem cells can thus be obtained, by separating other cells such as epithelial cells, dead cells, or red blood cells, or particulate matter, from the amniotic fluid sample source.
  • Cells of relatively high abundance in the amniotic fluid, compared to MSC:s, such epithelial cells, red blood cells, or particulate matter may be separated and removed from the amniotic fluid in order to increase the relative concentration of MSC:s in the obtained sample. At least the concentration of particulate matter or cells, such epithelial cells, or red blood cells, may be reduced by the separation to increase the relative concentration of MSC:s.
  • Amniotic stem cells of high quality can be obtained.
  • the desired stem cells are obtained with high yield and precision from the amniotic fluid which contains a vast range of different types of cells and particles as explained in the introductory part. Repeated and/or successive separation of the amniotic cells and particulate matter, as described further with reference to Figs. 4 - 6 below, may further increase the concentration of the cells of interest in the obtained cell sample.
  • the high yield provides also for utilizing amniotic fluid samples that would otherwise be discarded, since even samples having lower amounts of fluid, and thereby few extractable MSC:s, may be processed by the above described separation to increase the relative concentration of MSC:s.
  • EV extracellular vesicles
  • the amniotic cell separation apparatus 100 provides for minimizing exposure to contaminants and an efficient aseptic handling of the amniotic fluid. The risk of exposing the amniotic stem cells to contaminants, such as bacteria and viruses, is thus reduced. Exposure to oxygen is also minimized, which provides for reducing formation of oxygen free radicals which may have a negative impact the functioning of the stem cells. This facilitates obtaining amniotic cells which allows post-filtration processing at an improved quality standard. Flence, an aseptic pharmaceutical production process is facilitated. The preparation of e.g. surfactant molecules may be facilitated.
  • the amniotic cell separation apparatus 100 provides for improving the functioning of the amniotic stem cells, such as an improved engraftment phase following transplantation.
  • the controller 104 may be configured to drive the ultrasound transducer 103 with a frequency (f) to cause separation, along the transverse direction (r), between amniotic cells of the first morphology and amniotic cells of a second morphology being different from the first morphology.
  • the amniotic cells of the first and second morphologies may comprise respective first and second types of amniotic stem cells. Different types of amniotic stem cells may accordingly be separated and isolated based on their morphologies.
  • the amniotic cells of the first morphology may be Mesenchymal stem cells.
  • amniotic stem cells may be isolated from other cells or particulate matter in the amniotic fluid.
  • the controller 104 may thus be configured to drive the ultrasound transducer 103 with a frequency to cause separation between the stem cells and the other cells or particulate matter in the transverse direction (r) based on the different physical properties of the aforementioned cells and the particulate matter. It is conceivable that the frequency may be varied such that the fluidic trajectory of the amniotic stem cells to be isolated is diverted either to the walls of the fluidic channel forming the sample inlet 102, or to its center in the transverse direction (r). Fig.
  • stem cells of a first morphology (illustrated as spherical dots) are diverted to the bottom wall of the sample inlet 102, before being collected in separation channel 105.
  • the separation channel 105 may instead be arranged to collect the stem cells from such center position, while particulate matter or other cells which are not desirable are diverted to the walls of the fluidic channel before being collected to an outlet at either side of the central separation channel 105 (not shown).
  • amniotic stem cells to be isolated may be diverted as described, either to the walls or to the center, it is provided for movement of the other cells and particles in the opposite direction, due to having physical characteristics causing a different acoustophoretic contrast factor (f) as described above.
  • the controller 104 may thus be configured to drive the ultrasound transducer 103 with a frequency (f) to subject particulate matter and the amniotic cells of the first morphology to different acoustic forces along the transverse direction (r).
  • the particulate matter can thereby be separated and collected in an outlet channel 107 of the fluidic cavity 101.
  • the particulate matter may be collected in an outlet container 203 connected to the outlet channel 107, as schematically illustrated in Fig. 1.
  • the amniotic cell separation apparatus 100 may comprise a plurality of separation channels 105, 105’, 105”.
  • Fig. 4 is a schematic illustration of an amniotic cell separation apparatus 100 according to one example where a first separation channel 105 is branched into second and third separation channels 105’, 105”, along the longitudinal direction (L).
  • the controller 104 is configured to drive the ultrasound transducer 103a, 103b, with a frequency (f) to subject the amniotic cells of first and second morphologies to different acoustic forces along the transverse direction (r).
  • the first and second morphologies, and the respective acoustophoretic contrast factors (f) are different.
  • the amniotic cells of the first and second morphologies are in one example separated and collected into the respective separation channels 105’, 105”.
  • the controller 104 may be configured to drive the ultrasound transducer 103a with a frequency that force amniotic cells of the first and second morphologies into the first separation channel 105, while particulate matter is diverted into an outlet channel 107.
  • the controller 104 may be configured to drive the ultrasound transducer 103b with a frequency that subsequently force amniotic cells of the first morphology into the second separation channel 105’ and amniotic cells of the second morphology into the third separation channel 105”.
  • Fig. 4 illustrates first and second ultrasound transducer components 103a, 103b, along the longitudinal direction (L).
  • a single ultrasound transducer 103 may be arranged along the longitudinal direction (L) to drive the amniotic cells of the first and second morphologies and the particulate matter into different separation channels 105, 105’, 105”, as exemplified above.
  • the frequency is adapted to the acoustophoretic contrast factors (f) of the amniotic cells of the first and second morphologies and the particulate matter in the amniotic fluid.
  • the amniotic cell separation apparatus 100 thus provides for effectively separating and isolating the plurality of different cellular and particulate components contained in an amniotic fluid.
  • the amniotic fluid contains cells and particles having a wide spectrum of physical characteristics.
  • Having a plurality of separation channels 105, 105’, 105”, along the longitudinal direction (L) as described above provides for separation of a wide range of cells and particulate matter of different morphologies, sizes, densities etc.
  • epithelial cells are typically large compared to MSC:s, and red blood cells are small compared to MSC:s.
  • the MSC:s have a size in the range 10 - 20 mhi, and epithelial cells may be >50 mhi.
  • Red blood cells have a diameter of about 6 - 8 mhi.
  • the MSC:s and the red blood cells are in one example separated and collected into the respective separation channels 105’, 105”, while epithelial cells are diverted into an outlet channel 107 (Figs. 4 - 5).
  • the controller 104 may accordingly be configured to drive the ultrasound transducer 103a with a frequency that force MSC:s and red blood cells into a first separation channel 105, while epithelial cells are diverted into an outlet channel 107.
  • the controller 104 may be configured to drive the ultrasound transducer 103b with a frequency that subsequently force MSC:s into the second separation channel 105’ and red blood cells into the third separation channel 105”.
  • a gradual sorting of the multiple components of the amniotic fluid is thus provided along the longitudinal direction (L).
  • Separation techniques based on acoustic fields have been used previously on blood samples for diagnostic purposes. Blood is a significantly more homogenous liquid compared to amniotic fluid. A problem with previous techniques is thus inadequate capabilities in separating the multitude of different cells and particles in the amniotic fluid, having a wide range of different characteristics and sizes.
  • the amniotic fluid is thus more complex than blood, as it contains vernix, hair, surfactant and several other complex products and cells.
  • the amniotic fluid may even contain blood and meconium. Therefore, products in the amniotic fluid are more difficult to isolate than products in blood.
  • controller 104 configured to drive the ultrasound transducer 103, 103a, 103b, to force the particulate matter and the amniotic cells of the first and second morphologies in different transverse directions (r) into associated outlet and separation channels 107, 105, 105’, 105”, along the longitudinal direction (L) thus provides for an effective separation of the components of the amniotic fluid and isolation of the desired amniotic stem cells.
  • Fig. 5 is a schematic illustration of a further example of the amniotic cell separation apparatus 100.
  • the second separation channel 105’ in Fig. 5 is a continuous extension of the first separation channel 105. It is thus conceivable that the orientation of the separation channels 105, 105’, 105”, as well as the outlet channel 107 may optimized depending on the application and to what extent the trajectory of the particular amniotic cell or particulate matter may be diverted along the transverse direction (r).
  • the ultrasonic field created by the ultrasound transducer 103, 103a, 103b may be optimized to the different physical characteristics of the cells and particles of the amniotic fluid.
  • the ultrasound transducer may comprise a first transducer 103a and a second transducer 103b, as schematically illustrated in Figs. 3 - 5.
  • the first and second transducers 103a, 103b may be controlled independently by controller 104 to create an optimized acoustic field at the respective positions of the first and second transducers 103a, 103b.
  • the separation channel 105 may divert from an outlet channel 107 at a first branching portion 108a of the fluidic cavity 101.
  • the first transducer 103a may be arranged along the sample inlet 102.
  • the first transducer 103a may be arranged to extend in the longitudinal direction (L) to the position of the first branching portion 108a, as schematically illustrated in Fig. 4. This may provide for effectively diverting the amniotic cells and particulate matter into the different channels, i.e. the separation channel 105 and the outlet channel 107.
  • the second transducer 103b may be arranged to generate an acoustic wave in the separation channel 105 to separate amniotic cells in the transverse direction (r) at a second branching portion 108b arranged in the separation channel 105.
  • a second separation may be provided, e.g. of amniotic cells having different morphologies, into respective second and third separation channels 105’,
  • amniotic cell separation apparatus 100 thus provides for a high throughput in order to allow an efficient harvesting of the desired stem cells, even for large amniotic fluid samples, which in one example may be in the range 400 ml. to 2 L.
  • the ultrasound transducer may thus comprise a first transducer 103a and a second transducer 103b.
  • the controller 104 may be configured to drive the first and second transducers 103a, 103b, to generate at least two different acoustic waves in the fluidic cavity 101 along the longitudinal direction (L).
  • the first and second transducers 103a, 103b may be arranged along different channels, e.g. the sample inlet 102 or separation channels 105, 105’, 105”, such as illustrated in Figs. 4 and 5.
  • the first and second transducers 103a, 103b may in one example be arranged along the same channel, e.g. the sample inlet 102, such as schematically illustrated in Fig. 3.
  • At least two different acoustic waves may be generated along the aforementioned channels.
  • the generated pressure nodes and the associated acoustic force may thus be varied to optimize the manipulation of the amniotic cells and/or particles along said channels in the longitudinal direction (L). This provides for an effective manipulation of the amniotic cells and/or particles and optimization to their respective acoustophoretic contrast factors (f).
  • the ultrasound transducer 103, 103a, 103b may be arranged at an angle with respect to the longitudinal direction (L).
  • the ultrasound transducer 103 may be form a non-zero angle with respect to the longitudinal direction (L) (not shown). This provides in some examples for an advantageous orientation of the acoustic force and manipulation of the amniotic cells and/or particles in the desired direction.
  • the controller 104 may be configured to drive the ultrasound transducer 103, 103a, 103b, with a resonance frequency of the fluidic cavity 101.
  • a resonance frequency of the fluidic cavity 101 should be construed as a resonance frequency of any of the fluidic channels where the acoustic wave should be produced, i.e. the resonance frequency of the sample inlet 102, and/or separation channel 105, 105’, 105”. This provides for generating an acoustic standing wave in the related fluidic channel, resulting in pressure nodes and the exposing of the amniotic cells and particles to acoustic forces.
  • the controller 104 may be configured to drive the ultrasound transducer 103, 103a, 103b, with a frequency sweep (Af) around at least one resonance frequency (f) of the fluidic cavity 101.
  • the frequency may sweep from f - Af/2 to f + Af/2, where f is the resonance frequency of any of the sample inlet 102 and separation channel 105, 105’, 105”, and Af is the total range of the frequency sweep.
  • Driving the ultrasound transducer 103, 103a, 103b, with a frequency sweep around the resonance frequency provides in some examples for a more reliable and robust sorting of the amniotic cells. Variations or imperfections in the fluidic cavity 101 may lead to difficulties in finding an accurate resonance frequency.
  • any of the ultrasound transducers 103, 103a, 103b, described in relation to Figs. 1 - 7 may be configured to generate an acoustic wave with a frequency sweep around the resonance frequency.
  • the frequency may be varied from a frequency 20% below the resonance frequency to a frequency 20% above the resonance frequency.
  • the cross-sectional area of the first separation channel 105 and/or the second separation channel 105’ may be less than the cross-sectional area of the sample inlet 102 of the fluidic cavity 101. Having a gradually decreasing cross-sectional area of the fluidic channels of the fluidic cavity 101 when the amniotic fluid flows in the longitudinal direction (L) from the inlet 102 to the outlet 106 allows for accommodating and sorting a greater range of sizes of the amniotic cells and particles while maintaining high efficiency and throughput.
  • the sample inlet 102 may have a larger cross-section than the first separation channel 105 to facilitate having particulate matter of the amniotic fluid propagating in the sample inlet 102 before being diverted to the outlet channel 107.
  • the throughput of amniotic fluid may be increased.
  • the first separation channel 105 may have a larger cross-section than the second separation channel 105’. This allows for separating amniotic stem cells of interest into the second separation channel 105’ while maintaining a higher flow of the amniotic fluid through the first separation channel 105 which may also accommodate other non-desirable amniotic cells of larger size before being diverted to the third separation channel 105”.
  • the cross-sectional area of the first separation channel 105 and/or the second separation channel 105’ may be less than the cross-sectional area of the outlet channel 107 of the fluidic cavity 101. This provides maintaining a higher flow of the amniotic fluid through the fluidic cavity 101 , while separating the amniotic cells of interest.
  • the fluidic cavity 101 may comprise a feedback conduit 109 arranged for fluid communication between a separation channel 105 and the inlet 102, as schematically illustrated in Fig. 6.
  • An amount of amniotic fluid may be introduced into the sample inlet 102, e.g. via a valve at the inlet connector 112.
  • the controller 104 is configured to drive the ultrasound transducer 103a to generate an acoustic wave and an associated acoustic force to divert amniotic cells having a first morphology towards the separation channel 105, while particulate matter or remaining cells of a different morphology may be diverted towards an outlet channel 107.
  • the separation channel 105 and outlet channel 107 may have respective valves for controlling the flow therethrough.
  • the amniotic cells first diverted to the separation channel 105, or at least a fraction thereof, may be collected in the feedback conduit 109 and returned to the sample inlet 102 for a subsequent exposure to the acoustic waves and separation along the transverse direction (r) for a subsequent collection in the separation channel 105.
  • the amniotic cells to be isolated may thus be kept in the fluid loop provided by the feedback conduit 109, while particulate matter or non-desirable cells may be gradually removed via the outlet channel 107.
  • a valve in the separation channel 105 may be opened to retrieve the amniotic cells to be isolated from an outlet 106. This provides for a gradual sorting of the cells and particulate matter of the amniotic fluid.
  • a continuous refinement of the amniotic sample cells to be isolated may be provided.
  • a buffer solution 113 may be provided into the sample inlet 102 in one example to maintain a desired flow volume in the fluidic cavity 101. It is further conceivable that in another example the amniotic cells to be isolated are gradually retrieved from outlet channel 107, while the remaining amniotic cells and particulate matter is circulated in the feedback conduit 109 before being expelled through outlet 106.
  • the ultrasound transducer may comprise a first transducer 103a and a second transducer 103b.
  • the second transducer 103b may be arranged along the separation channel 105.
  • the controller 104 may be configured to drive the second ultrasound transducer 103b with a frequency to excite orientation of the amniotic cells of the first morphology along an axis, such as an axis parallel or perpendicular to the longitudinal direction (L).
  • the amniotic cells of the first morphology may thus be non-spherical (illustrated as elongated dots in Fig. 7).
  • the amniotic cell separation apparatus 100 may further comprise a sensor 110 arranged at the separation channel 105 to distinguish the amniotic cells of the first morphology from the amniotic fluid based on the excited orientation, as schematically illustrated in Fig. 7.
  • the amniotic cells of the first morphology may thus be oriented and distinguished for characterization purposes, e.g. of the performance of the sorting process.
  • the ratio between amniotic cells having the excited orientation and the remaining amniotic cells may e.g. be determined.
  • the sensor 110 may comprise and imaging sensor configured to retrieve image data of the amniotic cells diverted to the separation channel 105 to determine their orientation.
  • the frequency driving the second transducer 103b may be varied to excite orientation of the amniotic cells in different directions.
  • the change of orientation may be utilized for characterization of the morphology of the amniotic cells.
  • the sensor 110 comprise an electrical sensor configured characterize amniotic cells based on e.g. measurements of conductivity.
  • the amniotic cell separation apparatus 100 may comprise a pre-filter 111 arranged upstream of the sample inlet 102 to remove at least part of the particulate matter in the amniotic fluid, as schematically exemplified in Fig. 7.
  • the amniotic cell separation apparatus 100 comprises an electric field generator (not shown) being configured to exert the amniotic fluid to a non-uniform electric field when flowing along the sample inlet, to force cells and/or particles in the fluid in the transverse direction (r) due to dielectrophoresis.
  • the force acts on the cells and/or particles due to polarization effects in the cells and/or particles when exposed to the non-uniform electric field.
  • the cells and/or particles have different dielectric properties which will affect the amount of force and the amount of movement of the cells and/or particles in the transverse direction (r). Separation of the different cells and/or particles is thus provided, which may be used in combination with the above described acoustic force.
  • Fig. 8 shows a flow chart of a method 300 for separating amniotic cells in an amniotic fluid containing amniotic cells and particulate matter.
  • the method 200 comprises passing 301 the amniotic fluid through a sample inlet 102 of a fluid cavity 101.
  • the method 300 comprises generating 302 an acoustic wave in the fluidic cavity
  • the method thus provides for the advantageous benefits as described above for the amniotic cell separation apparatus 100 with reference to Figs. 1 - 7.
  • the method 300 provides for high yield and high- precision separation and isolation of desired amniotic cells such as stem cells with minimal alteration or destruction of cellular properties.
  • the method 300 provides for effective and sterile isolation of stem cells from an amniotic fluid to obtain amniotic cell samples of high quality.

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Abstract

An amniotic cell separation apparatus is disclosed comprising a fluidic cavity comprising an amniotic fluid sample inlet configured to connect to a sample source of amniotic fluid, the amniotic fluid containing amniotic cells and particulate matter, an ultrasound transducer arranged along the amniotic fluid sample inlet and being configured to generate an acoustic wave in the fluidic cavity, a controller connected to the ultrasound transducer, wherein the controller is configured to drive the ultrasound transducer with a frequency that generates said acoustic wave, whereby when an amniotic fluid flows in the sample inlet, the acoustic wave forces amniotic cells of a first morphology and/or the particulate matter along a transverse direction perpendicular to a longitudinal direction of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel of the fluidic cavity.

Description

AN AMNIOTIC CELL SEPARATING APPARATUS
Technical Field The present invention pertains to an apparatus for separating amniotic cells in an amniotic fluid and a related method.
Background Art
The amniotic fluid is the liquid surrounding and protecting the fetus during pregnancy. This fluid serves as a cushion for the growing fetus, but also serves to facilitate the exchange of nutrients, water, and biochemical products between mother and fetus. It is generated from maternal plasma, and passes through the fetal membranes by osmotic and hydrostatic forces. When fetal kidneys begin to function in about gestational week 16, fetal urine also contributes to the fluid. Amniotic fluid consists of water with electrolytes, but also contains proteins, carbohydrates, lipids, phospholipids, and urea. In addition to metabolic wastes, amniotic fluid also contains fetal cells and other materials chafed off the skin such as hair and vernix. The amniotic fluid contains a number of valuable cells, including Mesenchymal stem cells (MSC). The MSC:s possess the characteristics of self-renewing and differentiating into multiple cell types, including osteoblast, adipocyte, chondrocyte, myocyte, and fibroblast. The MSC:s can have a multilineage differentiation potential into bone, fat, and cartilage cells, and may thus be utilized for a variety of therapeutic applications, including personalized and regenerative medicine. The amniotic fluid also contains terminally differentiated cells with limited proliferation capacity. The MSC:s may be distinguished from other cells based on their morphology, or based on other characteristic such as genetic markers. It is thus possible to isolate stem cells with mesenchymal characteristics from the amniotic fluid. The morphology of a cell refers to parameters such as the shape, structure, or size of the cell. Morphologically, MSC:s are fibroblast like and spindle-shaped cells. Sorting based on morphology is traditionally accomplished by standard isolation techniques involving e.g. repeated centrifugation and washing of the cell sample.
The available approaches and apparatuses for collecting, extracting, and isolating the stem cells of interest are not entirely satisfactory, for example, in their safety, avoidance of contamination (e.g., air contamination) of collected material, cell yield, efficiency, and/or ability to avoid destruction of components. The quality of the stem cell samples may thus be negatively impacted, in particular in applications involving a large number of processing steps. The multitude of different cells and particles in the amniotic fluid, having a wide range of different characteristics and sizes, pose an additional challenge for the separation and isolation of the relevant stem cells.
Thus, while there is a need for separating and isolating multipotent and/or pluripotent stem cells, such as MSC:s, from more differentiated cells, for cell based therapeutic applications, the available methods and apparatuses are sub-optimal.
Summary
An objective is to at least partly overcome one or more of the above identified limitations of the prior art.
One objective is to provide an apparatus and method for high-yield, and high- precision, separation and isolation of desired amniotic cells such as stem cells with the potential for reprogramming, with minimal alteration or destruction of the cellular properties.
One or more of these objectives, and other objectives that may appear from the description below, are at least partly achieved by means of an apparatus and a method according to the independent claims, embodiments thereof being defined by the dependent claims.
According to a first aspect, an amniotic cell separation apparatus is provided comprising a fluidic cavity comprising an amniotic fluid sample inlet configured to connect to a sample source of amniotic fluid, the amniotic fluid containing amniotic cells and particulate matter, an ultrasound transducer arranged along the amniotic fluid sample inlet and being configured to generate an acoustic wave in the fluidic cavity, a controller connected to the ultrasound transducer, wherein the controller is configured to drive the ultrasound transducer with a frequency (f) that generates said acoustic wave, whereby when an amniotic fluid flows in the sample inlet, the acoustic wave forces amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel of the fluidic cavity, wherein the separation channel is connected to a first outlet connector to sealingly connect the separation channel to an amniotic cell receiving device.
According to a second aspect, a method for separating amniotic cells in an amniotic fluid containing amniotic cells and particulate matter is provided comprising passing the amniotic fluid through a sample inlet of a fluid cavity, and generating an acoustic wave in the fluidic cavity to force amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel of the fluidic cavity.
Further examples of the invention are defined in the dependent claims, wherein features for the first aspect may be implemented for the second aspect, and vice versa.
Some examples of the disclosure provide for an effective filtering of amniotic fluid.
Some examples of the disclosure provide for improved filtering of amniotic fluid containing a greater range in the size of particulate matter.
Some examples of the disclosure provide for obtaining stem cell samples of high quality.
Some examples of the disclosure provide for an efficient separation and isolation of stem cells of a desired morphology.
Some examples of the disclosure provide for a high-throughput isolation of stem cells from an amniotic fluid.
Some examples of the disclosure provide for a sterile isolation of stem cells from an amniotic fluid.
Some examples of the disclosure provide for a less complex apparatus for isolation of desired stem cells from of an amniotic fluid.
Some examples of the disclosure provide for a more compact apparatus for isolation of desired stem cells from of an amniotic fluid.
Some examples of the disclosure provide for obtaining an higher fraction of desired stem cells from a sample of amniotic fluid.
Still other objectives, features, aspects and advantages of the present disclosure will appear from the following detailed description, from the attached claims as well as from the drawings.
It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
Brief Description of Drawings
These and other aspects, features and advantages of which examples of the invention are capable of will be apparent and elucidated from the following description of examples of the present invention, reference being made to the accompanying drawings, in which;
Fig. 1 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 2 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 3 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 4 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 5 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 6 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 7 is a schematic illustration, in a cross-sectional view, of an amniotic cell separation apparatus, according to an example;
Fig. 8 is a flow chart of a method for separating amniotic cells in an amniotic fluid according to one example.
Detailed Description of Example Embodiments
Embodiments of the invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. The invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Throughout the description, the same reference numerals are used to identify corresponding elements.
Fig. 1 is a schematic illustration of an amniotic cell separation apparatus 100 according to one example. The amniotic cell separation apparatus 100 comprises a fluidic cavity 101. The fluidic cavity 101 comprises an amniotic fluid sample inlet 102 configured to connect to a sample source 201 of amniotic fluid. The apparatus 100 may comprise an inlet connector 112 arranged to sealingly connect the sample inlet 102 to the amniotic fluid sample source 201 . The amniotic fluid contains amniotic cells, such as stem cells, e.g. Mesenchymal stem cells (MSC). The amniotic fluid also contains terminally differentiated cells, e.g. keratinocytes, and other materials chafed off the skin such as hair and vernix. Material other than the amniotic cells are here referred to as particulate matter. The amniotic fluid thus generally contains a mixture of amniotic cells and particulate matter. The amniotic fluid may thus flow along the sample inlet 102 being a fluidic channel which extends along a longitudinal direction (L) as indicated in e.g. Fig. 1.
The amniotic cell separation apparatus 100 comprises an ultrasound transducer 103 arranged along the amniotic fluid sample inlet 102. The ultrasound transducer 103 is configured to generate an acoustic wave in the fluidic cavity 101. The amniotic cell separation apparatus 100 may comprise ultrasound transducer’s 103 arranged at opposite sides of the amniotic fluid sample inlet 102, along a transverse direction (r) perpendicular to the longitudinal direction (L) of the sample inlet 102, as schematically illustrated in the example of Fig. 1. It should be understood however that the amniotic cell separation apparatus 100 may comprise any number of ultrasound transducers 103, and that the position of the ultrasound transducer(s) 103 relative to the sample inlet 102 can be varied to optimize the characteristics of the acoustic wave in the fluidic cavity 101 and in the sample inlet 102 thereof. Figs. 3 - 5 are schematic illustrations of further examples of the amniotic cell separation apparatus 100 which will be described further below.
The amniotic cell separation apparatus 100 comprises a controller 104 connected to the ultrasound transducer 103. The controller 104 is configured to drive the ultrasound transducer 103 with a frequency (f) that generates the aforementioned acoustic wave. When an amniotic fluid flows along the sample inlet 102, i.e. along the longitudinal direction (L) of the sample inlet 102, the acoustic wave forces amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r), so that the amniotic cells of the first morphology are separated and collected in a separation channel 105 of the fluidic cavity 101 . The transverse direction (r) is perpendicular to the longitudinal direction (L) of the sample inlet 102.
The acoustic wave, or acoustic field, generated by the ultrasound transducer 103 creates acoustic pressure nodes across the width of the sample inlet 102, in the transverse direction (r), because of the acoustic vibrations. The amniotic cells and the particulate matter will be exposed to a net force due to the pressure nodes which is dependent on the physical properties of the amniotic cells and particulate matter relative to the surrounding medium in which the amniotic cells and particulate matter are suspended. The force exerted onto the amniotic cells or particles is also referred to as the acoustic radiation force. The direction of the force is dependent on the relationship between the physical properties of the amniotic cells or particles and the surrounding medium. The relationship can be expressed in the form of an acoustophoretic contrast factor (f). The factor f is determined by the relative compressibilities and the densities of the medium and the cell/particle. Amniotic cells having different morphologies, i.e. different physical properties, can thus have different associated acoustophoretic contrast factors (f). The direction of movement of the amniotic cells relative to the pressure nodes which extend in the transverse direction (r) thereby depends on the acoustophoretic contrast factor (f) of the particular amniotic cell. The different amniotic cells may thus be displaced in different directions along the transverse direction (r). For example, amniotic cells having a positive acoustophoretic contrast factor (f > 0) may be displaced in a first direction such as towards the transverse direction (r) illustrated in Fig. 2 (vertically upwards in Fig. 2), while amniotic cells having a negative acoustophoretic contrast factor (f < 0) may be displaced in a second direction, such as opposite the first direction (vertically downwards in Fig. 2). The schematic illustration of Fig. 2 thus shows an example where cells of different morphologies are separated along the transverse direction (r) as the amniotic fluid flows along the longitudinal direction (L).
Amniotic cells of a first morphology, such as MSC:s, are separated and collected in a separation channel 105 of the fluidic cavity 101 . T urning again to Fig. 1 , the separation channel 105 is connected to a first outlet connector 106 to sealingly connect the separation channel 105 to an amniotic cell-receiving device 202, where the amniotic cells of the first morphology may be collected. The amniotic cells of the first morphology may in one example comprise extracellular vesicles (EV’s). Extracellular vesicles are present in the amniotic fluid as lipid bilayer-delimited particles that are naturally released from a cell, and may have desirable properties for therapeutic purposes, such as delivering nucleic acids or other cargo to diseased tissue.
Thus, amniotic cells and particles having different morphologies and characteristics are displaced different amounts in the transverse direction (r) to control the respective flow trajectories in the fluidic cavity 101 , for separation and isolation of the desired amniotic cells. This provides for an effective separation of stem cells of interest, such as MSC:s, from an amniotic fluid sample source. The amniotic fluid may contain a relatively low concentration of stem cells, such as MSC:s. For example, the concentration may be 1 cell/mL amniotic fluid for the MSC:s. The separation allows for increasing the aforementioned concentration in the cell sample obtained by the amniotic cell separation apparatus 100. Stem cell samples of a desired concentration of stem cells can thus be obtained, by separating other cells such as epithelial cells, dead cells, or red blood cells, or particulate matter, from the amniotic fluid sample source. Cells of relatively high abundance in the amniotic fluid, compared to MSC:s, such epithelial cells, red blood cells, or particulate matter, may be separated and removed from the amniotic fluid in order to increase the relative concentration of MSC:s in the obtained sample. At least the concentration of particulate matter or cells, such epithelial cells, or red blood cells, may be reduced by the separation to increase the relative concentration of MSC:s.
Amniotic stem cells of high quality can be obtained. The desired stem cells are obtained with high yield and precision from the amniotic fluid which contains a vast range of different types of cells and particles as explained in the introductory part. Repeated and/or successive separation of the amniotic cells and particulate matter, as described further with reference to Figs. 4 - 6 below, may further increase the concentration of the cells of interest in the obtained cell sample. The high yield provides also for utilizing amniotic fluid samples that would otherwise be discarded, since even samples having lower amounts of fluid, and thereby few extractable MSC:s, may be processed by the above described separation to increase the relative concentration of MSC:s.
The separation of extracellular vesicles (EV’s) may be advantageous in some applications due to a higher abundance in the amniotic fluid.
Further, the amniotic cell separation apparatus 100 provides for minimizing exposure to contaminants and an efficient aseptic handling of the amniotic fluid. The risk of exposing the amniotic stem cells to contaminants, such as bacteria and viruses, is thus reduced. Exposure to oxygen is also minimized, which provides for reducing formation of oxygen free radicals which may have a negative impact the functioning of the stem cells. This facilitates obtaining amniotic cells which allows post-filtration processing at an improved quality standard. Flence, an aseptic pharmaceutical production process is facilitated. The preparation of e.g. surfactant molecules may be facilitated. The amniotic cell separation apparatus 100 provides for improving the functioning of the amniotic stem cells, such as an improved engraftment phase following transplantation.
In one example the controller 104 may be configured to drive the ultrasound transducer 103 with a frequency (f) to cause separation, along the transverse direction (r), between amniotic cells of the first morphology and amniotic cells of a second morphology being different from the first morphology. The amniotic cells of the first and second morphologies may comprise respective first and second types of amniotic stem cells. Different types of amniotic stem cells may accordingly be separated and isolated based on their morphologies. The amniotic cells of the first morphology may be Mesenchymal stem cells.
In one example amniotic stem cells may be isolated from other cells or particulate matter in the amniotic fluid. The controller 104 may thus be configured to drive the ultrasound transducer 103 with a frequency to cause separation between the stem cells and the other cells or particulate matter in the transverse direction (r) based on the different physical properties of the aforementioned cells and the particulate matter. It is conceivable that the frequency may be varied such that the fluidic trajectory of the amniotic stem cells to be isolated is diverted either to the walls of the fluidic channel forming the sample inlet 102, or to its center in the transverse direction (r). Fig. 2 shows an example where stem cells of a first morphology (illustrated as spherical dots) are diverted to the bottom wall of the sample inlet 102, before being collected in separation channel 105. In case the fluidic trajectory of the stem cells to be isolated is directed towards the center of the sample inlet 102 channel it should be understood that the separation channel 105 may instead be arranged to collect the stem cells from such center position, while particulate matter or other cells which are not desirable are diverted to the walls of the fluidic channel before being collected to an outlet at either side of the central separation channel 105 (not shown). While the amniotic stem cells to be isolated may be diverted as described, either to the walls or to the center, it is provided for movement of the other cells and particles in the opposite direction, due to having physical characteristics causing a different acoustophoretic contrast factor (f) as described above.
The controller 104 may thus be configured to drive the ultrasound transducer 103 with a frequency (f) to subject particulate matter and the amniotic cells of the first morphology to different acoustic forces along the transverse direction (r). The particulate matter can thereby be separated and collected in an outlet channel 107 of the fluidic cavity 101. The particulate matter may be collected in an outlet container 203 connected to the outlet channel 107, as schematically illustrated in Fig. 1.
The amniotic cell separation apparatus 100 may comprise a plurality of separation channels 105, 105’, 105”. Fig. 4 is a schematic illustration of an amniotic cell separation apparatus 100 according to one example where a first separation channel 105 is branched into second and third separation channels 105’, 105”, along the longitudinal direction (L). The controller 104 is configured to drive the ultrasound transducer 103a, 103b, with a frequency (f) to subject the amniotic cells of first and second morphologies to different acoustic forces along the transverse direction (r). The first and second morphologies, and the respective acoustophoretic contrast factors (f) are different. The amniotic cells of the first and second morphologies are in one example separated and collected into the respective separation channels 105’, 105”. E.g. the controller 104 may be configured to drive the ultrasound transducer 103a with a frequency that force amniotic cells of the first and second morphologies into the first separation channel 105, while particulate matter is diverted into an outlet channel 107. The controller 104 may be configured to drive the ultrasound transducer 103b with a frequency that subsequently force amniotic cells of the first morphology into the second separation channel 105’ and amniotic cells of the second morphology into the third separation channel 105”. Fig. 4 illustrates first and second ultrasound transducer components 103a, 103b, along the longitudinal direction (L). It should be understood however that in other examples a single ultrasound transducer 103 may be arranged along the longitudinal direction (L) to drive the amniotic cells of the first and second morphologies and the particulate matter into different separation channels 105, 105’, 105”, as exemplified above. The frequency is adapted to the acoustophoretic contrast factors (f) of the amniotic cells of the first and second morphologies and the particulate matter in the amniotic fluid. The amniotic cell separation apparatus 100 thus provides for effectively separating and isolating the plurality of different cellular and particulate components contained in an amniotic fluid. The amniotic fluid contains cells and particles having a wide spectrum of physical characteristics. Having a plurality of separation channels 105, 105’, 105”, along the longitudinal direction (L) as described above provides for separation of a wide range of cells and particulate matter of different morphologies, sizes, densities etc. For example, epithelial cells are typically large compared to MSC:s, and red blood cells are small compared to MSC:s. In one example the MSC:s have a size in the range 10 - 20 mhi, and epithelial cells may be >50 mhi. Red blood cells have a diameter of about 6 - 8 mhi.
The MSC:s and the red blood cells are in one example separated and collected into the respective separation channels 105’, 105”, while epithelial cells are diverted into an outlet channel 107 (Figs. 4 - 5). The controller 104 may accordingly be configured to drive the ultrasound transducer 103a with a frequency that force MSC:s and red blood cells into a first separation channel 105, while epithelial cells are diverted into an outlet channel 107. The controller 104 may be configured to drive the ultrasound transducer 103b with a frequency that subsequently force MSC:s into the second separation channel 105’ and red blood cells into the third separation channel 105”.
A gradual sorting of the multiple components of the amniotic fluid is thus provided along the longitudinal direction (L). Separation techniques based on acoustic fields have been used previously on blood samples for diagnostic purposes. Blood is a significantly more homogenous liquid compared to amniotic fluid. A problem with previous techniques is thus inadequate capabilities in separating the multitude of different cells and particles in the amniotic fluid, having a wide range of different characteristics and sizes.
The amniotic fluid is thus more complex than blood, as it contains vernix, hair, surfactant and several other complex products and cells. The amniotic fluid may even contain blood and meconium. Therefore, products in the amniotic fluid are more difficult to isolate than products in blood.
Having a controller 104 configured to drive the ultrasound transducer 103, 103a, 103b, to force the particulate matter and the amniotic cells of the first and second morphologies in different transverse directions (r) into associated outlet and separation channels 107, 105, 105’, 105”, along the longitudinal direction (L) thus provides for an effective separation of the components of the amniotic fluid and isolation of the desired amniotic stem cells.
Fig. 5 is a schematic illustration of a further example of the amniotic cell separation apparatus 100. Rather than having separation channels such as the second and third separation channels 105’, 105”, branching in opposite directions as exemplified at branching portion 108b in Fig. 4, the second separation channel 105’ in Fig. 5 is a continuous extension of the first separation channel 105. It is thus conceivable that the orientation of the separation channels 105, 105’, 105”, as well as the outlet channel 107 may optimized depending on the application and to what extent the trajectory of the particular amniotic cell or particulate matter may be diverted along the transverse direction (r).
The ultrasonic field created by the ultrasound transducer 103, 103a, 103b, may be optimized to the different physical characteristics of the cells and particles of the amniotic fluid. In one example, the ultrasound transducer may comprise a first transducer 103a and a second transducer 103b, as schematically illustrated in Figs. 3 - 5. The first and second transducers 103a, 103b, may be controlled independently by controller 104 to create an optimized acoustic field at the respective positions of the first and second transducers 103a, 103b. Turning again to Fig. 4, the separation channel 105 may divert from an outlet channel 107 at a first branching portion 108a of the fluidic cavity 101. The first transducer 103a may be arranged along the sample inlet 102. In one example, the first transducer 103a may be arranged to extend in the longitudinal direction (L) to the position of the first branching portion 108a, as schematically illustrated in Fig. 4. This may provide for effectively diverting the amniotic cells and particulate matter into the different channels, i.e. the separation channel 105 and the outlet channel 107. The second transducer 103b may be arranged to generate an acoustic wave in the separation channel 105 to separate amniotic cells in the transverse direction (r) at a second branching portion 108b arranged in the separation channel 105. Thus, as elucidated above, a second separation may be provided, e.g. of amniotic cells having different morphologies, into respective second and third separation channels 105’,
105”. Such gradual sorting provides for sorting of a greater range of physically different amniotic cells and particles in the amniotic fluid. This provides also for an increased throughput of the sorting. Previous acoustic sorting techniques are concerned with diagnosis of blood which is on the contrary not bound by high throughput. The amniotic cell separation apparatus 100 thus provides for a high throughput in order to allow an efficient harvesting of the desired stem cells, even for large amniotic fluid samples, which in one example may be in the range 400 ml. to 2 L.
The ultrasound transducer may thus comprise a first transducer 103a and a second transducer 103b. The controller 104 may be configured to drive the first and second transducers 103a, 103b, to generate at least two different acoustic waves in the fluidic cavity 101 along the longitudinal direction (L). The first and second transducers 103a, 103b, may be arranged along different channels, e.g. the sample inlet 102 or separation channels 105, 105’, 105”, such as illustrated in Figs. 4 and 5. The first and second transducers 103a, 103b, may in one example be arranged along the same channel, e.g. the sample inlet 102, such as schematically illustrated in Fig. 3. Regardless, at least two different acoustic waves may be generated along the aforementioned channels. The generated pressure nodes and the associated acoustic force may thus be varied to optimize the manipulation of the amniotic cells and/or particles along said channels in the longitudinal direction (L). This provides for an effective manipulation of the amniotic cells and/or particles and optimization to their respective acoustophoretic contrast factors (f).
In one example the ultrasound transducer 103, 103a, 103b, may be arranged at an angle with respect to the longitudinal direction (L). E.g. instead of having a parallel orientation of the ultrasound transducer 103 with respect to the longitudinal direction (L) in Fig. 1 (i.e. an angle of zero with respect to the longitudinal direction (L)), the ultrasound transducer 103 may be form a non-zero angle with respect to the longitudinal direction (L) (not shown). This provides in some examples for an advantageous orientation of the acoustic force and manipulation of the amniotic cells and/or particles in the desired direction.
In one example the controller 104 may be configured to drive the ultrasound transducer 103, 103a, 103b, with a resonance frequency of the fluidic cavity 101. A resonance frequency of the fluidic cavity 101 should be construed as a resonance frequency of any of the fluidic channels where the acoustic wave should be produced, i.e. the resonance frequency of the sample inlet 102, and/or separation channel 105, 105’, 105”. This provides for generating an acoustic standing wave in the related fluidic channel, resulting in pressure nodes and the exposing of the amniotic cells and particles to acoustic forces.
In one example the controller 104 may be configured to drive the ultrasound transducer 103, 103a, 103b, with a frequency sweep (Af) around at least one resonance frequency (f) of the fluidic cavity 101. For example, the frequency may sweep from f - Af/2 to f + Af/2, where f is the resonance frequency of any of the sample inlet 102 and separation channel 105, 105’, 105”, and Af is the total range of the frequency sweep. Driving the ultrasound transducer 103, 103a, 103b, with a frequency sweep around the resonance frequency provides in some examples for a more reliable and robust sorting of the amniotic cells. Variations or imperfections in the fluidic cavity 101 may lead to difficulties in finding an accurate resonance frequency. This may in particular be the case if the fluidic cavity 101 is manufactured from a polymer, which can be advantageous with respect to mass production, manufacturing costs, and sterile use. Utilizing a frequency sweep as described provides thus takes into account such variations and the associated variations in the optimal frequency for driving the acoustic field. Any of the ultrasound transducers 103, 103a, 103b, described in relation to Figs. 1 - 7 may be configured to generate an acoustic wave with a frequency sweep around the resonance frequency. In one example, the frequency may be varied from a frequency 20% below the resonance frequency to a frequency 20% above the resonance frequency.
Turning again to the example of Fig. 5, showing the second branching portion 108b connecting to a second separation channel 105’. The cross-sectional area of the first separation channel 105 and/or the second separation channel 105’ may be less than the cross-sectional area of the sample inlet 102 of the fluidic cavity 101. Having a gradually decreasing cross-sectional area of the fluidic channels of the fluidic cavity 101 when the amniotic fluid flows in the longitudinal direction (L) from the inlet 102 to the outlet 106 allows for accommodating and sorting a greater range of sizes of the amniotic cells and particles while maintaining high efficiency and throughput. For example, the sample inlet 102 may have a larger cross-section than the first separation channel 105 to facilitate having particulate matter of the amniotic fluid propagating in the sample inlet 102 before being diverted to the outlet channel 107. The throughput of amniotic fluid may be increased. In another example, the first separation channel 105 may have a larger cross-section than the second separation channel 105’. This allows for separating amniotic stem cells of interest into the second separation channel 105’ while maintaining a higher flow of the amniotic fluid through the first separation channel 105 which may also accommodate other non-desirable amniotic cells of larger size before being diverted to the third separation channel 105”.
The cross-sectional area of the first separation channel 105 and/or the second separation channel 105’ may be less than the cross-sectional area of the outlet channel 107 of the fluidic cavity 101. This provides maintaining a higher flow of the amniotic fluid through the fluidic cavity 101 , while separating the amniotic cells of interest.
The fluidic cavity 101 may comprise a feedback conduit 109 arranged for fluid communication between a separation channel 105 and the inlet 102, as schematically illustrated in Fig. 6. An amount of amniotic fluid may be introduced into the sample inlet 102, e.g. via a valve at the inlet connector 112. The controller 104 is configured to drive the ultrasound transducer 103a to generate an acoustic wave and an associated acoustic force to divert amniotic cells having a first morphology towards the separation channel 105, while particulate matter or remaining cells of a different morphology may be diverted towards an outlet channel 107. The separation channel 105 and outlet channel 107 may have respective valves for controlling the flow therethrough. The amniotic cells first diverted to the separation channel 105, or at least a fraction thereof, may be collected in the feedback conduit 109 and returned to the sample inlet 102 for a subsequent exposure to the acoustic waves and separation along the transverse direction (r) for a subsequent collection in the separation channel 105. The amniotic cells to be isolated may thus be kept in the fluid loop provided by the feedback conduit 109, while particulate matter or non-desirable cells may be gradually removed via the outlet channel 107. A valve in the separation channel 105 may be opened to retrieve the amniotic cells to be isolated from an outlet 106. This provides for a gradual sorting of the cells and particulate matter of the amniotic fluid. A continuous refinement of the amniotic sample cells to be isolated may be provided. A buffer solution 113 may be provided into the sample inlet 102 in one example to maintain a desired flow volume in the fluidic cavity 101. It is further conceivable that in another example the amniotic cells to be isolated are gradually retrieved from outlet channel 107, while the remaining amniotic cells and particulate matter is circulated in the feedback conduit 109 before being expelled through outlet 106.
Turning to the example of Fig. 7, the ultrasound transducer may comprise a first transducer 103a and a second transducer 103b. The second transducer 103b may be arranged along the separation channel 105. The controller 104 may be configured to drive the second ultrasound transducer 103b with a frequency to excite orientation of the amniotic cells of the first morphology along an axis, such as an axis parallel or perpendicular to the longitudinal direction (L). The amniotic cells of the first morphology may thus be non-spherical (illustrated as elongated dots in Fig. 7). The amniotic cell separation apparatus 100 may further comprise a sensor 110 arranged at the separation channel 105 to distinguish the amniotic cells of the first morphology from the amniotic fluid based on the excited orientation, as schematically illustrated in Fig. 7.
The amniotic cells of the first morphology may thus be oriented and distinguished for characterization purposes, e.g. of the performance of the sorting process. The ratio between amniotic cells having the excited orientation and the remaining amniotic cells may e.g. be determined. The sensor 110 may comprise and imaging sensor configured to retrieve image data of the amniotic cells diverted to the separation channel 105 to determine their orientation. In one example the frequency driving the second transducer 103b may be varied to excite orientation of the amniotic cells in different directions. The change of orientation may be utilized for characterization of the morphology of the amniotic cells. It is also conceivable that the sensor 110 comprise an electrical sensor configured characterize amniotic cells based on e.g. measurements of conductivity.
The amniotic cell separation apparatus 100 may comprise a pre-filter 111 arranged upstream of the sample inlet 102 to remove at least part of the particulate matter in the amniotic fluid, as schematically exemplified in Fig. 7.
In one example the amniotic cell separation apparatus 100 comprises an electric field generator (not shown) being configured to exert the amniotic fluid to a non-uniform electric field when flowing along the sample inlet, to force cells and/or particles in the fluid in the transverse direction (r) due to dielectrophoresis. The force acts on the cells and/or particles due to polarization effects in the cells and/or particles when exposed to the non-uniform electric field. The cells and/or particles have different dielectric properties which will affect the amount of force and the amount of movement of the cells and/or particles in the transverse direction (r). Separation of the different cells and/or particles is thus provided, which may be used in combination with the above described acoustic force.
Fig. 8 shows a flow chart of a method 300 for separating amniotic cells in an amniotic fluid containing amniotic cells and particulate matter. The method 200 comprises passing 301 the amniotic fluid through a sample inlet 102 of a fluid cavity 101. The method 300 comprises generating 302 an acoustic wave in the fluidic cavity
101 to force 303 amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet
102 so that the amniotic cells of the first morphology are separated are collected in a separation channel 105 of the fluidic cavity 101 . The method thus provides for the advantageous benefits as described above for the amniotic cell separation apparatus 100 with reference to Figs. 1 - 7. The method 300 provides for high yield and high- precision separation and isolation of desired amniotic cells such as stem cells with minimal alteration or destruction of cellular properties. The method 300 provides for effective and sterile isolation of stem cells from an amniotic fluid to obtain amniotic cell samples of high quality.
From the description above follows that, although various embodiments of the invention have been described and shown, the invention is not restricted thereto, but may also be embodied in other ways within the scope of the subject-matter defined in the following claims.

Claims

Claims
1 . An amniotic cell separation apparatus (100) comprising a fluidic cavity (101) comprising an amniotic fluid sample inlet (102) configured to connect to a sample source (201 ) of amniotic fluid, the amniotic fluid containing amniotic cells and particulate matter, an ultrasound transducer (103, 103a, 103b) arranged along the amniotic fluid sample inlet and being configured to generate an acoustic wave in the fluidic cavity, a controller (104) connected to the ultrasound transducer, wherein the controller is configured to drive the ultrasound transducer with a frequency (f) that generates said acoustic wave, whereby when an amniotic fluid flows in the sample inlet, the acoustic wave forces amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel (105) of the fluidic cavity, wherein the separation channel is connected to a first outlet connector (106) to sealingly connect the separation channel to an amniotic cell-receiving device (202).
2. Amniotic cell separation apparatus according to claim 1 , wherein the controller is configured to drive the ultrasound transducer with a frequency (f) to cause separation, along the transverse direction, between amniotic cells of the first morphology and amniotic cells of a second morphology being different from the first morphology.
3. Amniotic cell separation apparatus according to claim 1 or 2, wherein amniotic cells of the first morphology comprise Mesenchymal stem cells.
4. Amniotic cell separation apparatus according to claim 1 or 2, wherein amniotic cells of the first morphology comprise extracellular vesicles.
5. Amniotic cell separation apparatus according to claim 2, comprising a plurality of separation channels (105, 105’, 105”), wherein the controller is configured to drive the ultrasound transducer with a frequency (f) to subject the amniotic cells of the first and second morphologies to different acoustic forces along the transverse direction, whereby the amniotic cells of the first and second morphologies are separated and collected into respective separation channels.
6. Amniotic cell separation apparatus according to any of claims 1 - 5, wherein the controller is configured to drive the ultrasound transducer with a frequency (f) to subject the particulate matter and the amniotic cells of the first morphology to different acoustic forces along the transverse direction, whereby the particulate matter is separated and collected in an outlet channel (107) of the fluidic cavity.
7. Amniotic cell separation apparatus according to any of claims 1 - 6, wherein the controller is configured to drive the ultrasound transducer with a frequency sweep Af around at least one resonance frequency of the fluidic cavity.
8. Amniotic cell separation apparatus according to any of claims 1 - 7, wherein the ultrasound transducer comprises a first transducer (103a) and a second transducer (103b), wherein the controller is configured to drive the first and second transducers to generate at least two different acoustic waves in the fluidic cavity along the longitudinal direction (L).
9. Amniotic cell separation apparatus according to any of claims 1 - 8, wherein the ultrasound transducer comprises a first transducer (103a) and a second transducer (103b), the separation channel (105) diverts from an outlet channel (107) at a first branching portion (108a) of the fluidic cavity, the second transducer (103b) is arranged to generate an acoustic wave in the separation channel (105) to separate amniotic cells in the transverse direction at a second branching portion (108b) arranged in the separation channel.
10. Amniotic cell separation apparatus according to claim 9, wherein the separation channel is a first separation channel, and wherein the second branching portion (108b) connects to a second separation channel (105’), wherein the cross- sectional area of the first and/or second separation channel is less than the cross- sectional area of the sample inlet and/or an outlet channel (107) of the fluidic cavity.
11. Amniotic cell separation apparatus according to any of claims 1 - 10, wherein the fluidic cavity comprises a feedback conduit (109) arranged for fluid communication between the separation channel and the inlet (102), whereby at least a fraction of amniotic cells first separated in the separation channel are collected in the feedback conduit for a subsequent exposure to the acoustic waves and separation along the transverse direction (r) for a subsequent collection in the separation channel.
12. Amniotic cell separation apparatus according to any of claims 1 - 11 , wherein the ultrasound transducer comprises a first transducer (103a) and a second transducer (103b), the second transducer being arranged along the separation channel, wherein the controller is configured to drive the second ultrasound transducer with a frequency (f) to excite orientation of the amniotic cells of the first morphology along an axis, the amniotic cells of the first morphology being non-spherical, wherein the amniotic cell separation apparatus further comprises a sensor (110) to distinguish the amniotic cells of the first morphology from the amniotic fluid based on the orientation.
13. Amniotic cell separation apparatus according to any of claims 1 - 12, comprising a pre-filter (111) arranged upstream of the sample inlet to remove at least part of the particulate matter in the amniotic fluid.
14. A method (300) for separating amniotic cells in an amniotic fluid containing amniotic cells and particulate matter, comprising passing (301) the amniotic fluid through a sample inlet (102) of a fluid cavity (101), and generating (302) an acoustic wave in the fluidic cavity to force (303) amniotic cells of a first morphology and/or the particulate matter along a transverse direction (r) perpendicular to a longitudinal direction (L) of the sample inlet so that the amniotic cells of the first morphology are separated and collected in a separation channel (105) of the fluidic cavity.
PCT/SE2022/050111 2021-02-09 2022-02-02 An amniotic cell separating apparatus WO2022173345A1 (en)

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